Tetravalent anti-psgl-1 antibodies and uses thereof

ABSTRACT

Provided herein are tetravalent antibodies that specifically bind to human PSGL-1. Unlike bivalent antibodies, these tetravalent antibodies contain a dimer of two monomers, with each monomer comprising two light chain variable (VL) domains and two heavy chain variable (VH) domains. This format allows for cross-linker/FcR-expressing cell-independent tetravalent antibodies against PSGL-1 that show enhanced efficacy as compared to bivalent PSGL-1 antibodies. These tetravalent antibodies can be used in a variety of diagnostic and therapeutic methods, including without limitation treating T-cell mediated inflammatory diseases, transplantations, and transfusions.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the priority benefit of U.S. ProvisionalApplication Ser. No. 62/276,806, filed Jan. 8, 2016, which isincorporated herein by reference in its entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 606592001300SEQLIST.TXT,date recorded: Jan. 3, 2017, size: 70 KB).

FIELD

Provided herein are tetravalent antibodies that specifically bind tohuman P-selectin glycoprotein ligand-1 (PSGL-1), as well aspolynucleotides, vectors, host cells, methods, pharmaceuticalcompositions, kits, and uses related thereto. These tetravalentantibodies may find use in a variety of diagnostic and therapeuticmethods, including without limitation treating T-cell mediatedinflammatory diseases, transplantations, and transfusions.

BACKGROUND

Inflammatory responses to infection or injury are initiated by theadherence of leukocytes to the vascular wall (McEver et al, 1997, J.Clin. Invest., 100 (3): 485-492). Selectin represents a family ofglycoproteins which mediate the first leukocyte-endothelial cell andleukocyte-platelet interactions during inflammation. The selectinfamily, which consists of L-selectin, E-selectin, and P-selectin,comprises an NH₂-terminal lectin domain, followed by an EGF-like domain,a series of consensus repeats, a transmembrane domain, and a shortcytoplasmic tail. The lectin domains of selectins interact with specificglycoconjugate ligands in order to facilitate cell adhesion. L-selectin,expressed on most leukocytes, binds to ligands on some endothelial cellsand other leukocytes. E-selectin, expressed on cytokine activatedendothelial cells, binds to ligands on most leukocytes. P-selectin,expressed on activated platelets and endothelial cells, also binds toligands on most leukocytes.

P-selectin glycoprotein ligand-1 (“PSGL-1”), also known as SELPLG orCD162 (cluster of differentiation 162) is a human mucin-typeglycoprotein ligand for all three selectins (Constantin, Gabriela, 2004,Drug News Perspect., 17(9): 579-585; McEver et al., 1997, J. Clin.Invest., 100 (3): 485-492). PSGL-1 is a disulfide-bonded homodimer withtwo 120-kD subunits and is expressed on the surface of monocytes,lymphocytes, granulocytes, and in some CD34⁺ stem cells. PSGL-1 islikely to contribute to pathological leukocyte recruitment in manyinflammatory disorders since it facilitates the adhesive interactions ofselectins. In addition, PSGL-1 is shown to have a unique regulatory rolein T cells. Mice deficient in PSGL-1 show enhanced proliferativeresponses and autoimmunity, suggesting that PSGL-1 plays an importantrole in down-regulating T cell responses (Krystle M. et al. J. Immunol.2012; 188:1638-1646. Urzainqui et al. Ann Rheum Dis 2013; 71:650;Pérez-Frías A, et al. Arthritis Rheumatol. 2014 November;66(11):3178-89.; Angiari et al. J Immunol. 2013; 191(11):5489-500).

Several anti-PSGL-1 antibodies have been developed (see, e.g.,International Application Pub. Nos. WO 2005/110475, WO 2003/013603, andWO 2012/174001; Constantin, Gabriela, 2004, Drug News Perspect., 17(9):579-585, Chen et al. Blood. 2004; 104(10):3233-42, Huang et al, Eur JImmunol. 2005; 35(7):2239-49; and U.S. Pat. No. 7,604,800). Some of theexisting agonistic PSGL-1 antibodies preferentially induce apoptosis oflate-stage activated T cells but not other PSGL-1-expressing cells; suchantibodies may therefore be useful as anti-inflammatory therapeutics, orfor use in transplantations and/or transfusions. However, a need existsfor improved anti-PSGL-1 antibodies with greater in vivo efficacy thanexisting antibodies.

All publications, patents, and patent applications cited herein arehereby incorporated by reference in their entirety for all purposes.

BRIEF SUMMARY

To meet this need, provided herein are tetravalent antibodies thatspecifically bind to human PSGL-1, as well as polynucleotides, vectors,host cells, methods, pharmaceutical compositions, kits, and uses relatedthereto. The present disclosure demonstrates that tetravalent antibodiesthat specifically bind to human PSGL-1 have greater potency and efficacythan conventional (e.g., bivalent) anti-PSGL-1 antibodies. As such,these tetravalent antibodies may find use, inter alia, in diagnosticand/or therapeutic methods, uses, and compositions related to T-cellfunction, such as in treating T-cell mediated inflammatory diseases,transfusions, and/or transplantations.

Accordingly, in one aspect, provided herein is a tetravalent antibodythat specifically binds to human PSGL-1, the tetravalent antibodycomprising a dimer of two monomers, wherein each monomer of the dimercomprises a single-chain polypeptide comprising: (a) two light chainvariable (VL) domains, wherein each of the two VL domains comprises aCDR-L1, a CDR-L2, and a CDR-L3; (b) two heavy chain variable (VH)domains, wherein each of the two VH domains comprises a CDR-H1, aCDR-H2, and a CDR-H3; and (c) an antibody Fc domain, wherein each of thetwo VL domains forms a VH-VL binding unit with a corresponding VH domainof the two VH domains, and wherein each of the two VH-VL binding unitsis specific for human PSGL-1. In some embodiments, at least one of thetwo VH domains comprises: (i) a CDR-H1 comprising the amino acidsequence of SEQ ID NO:17; (ii) a CDR-H2 comprising the amino acidsequence of SEQ ID NO:18; and (iii) a CDR-H3 comprising the amino acidsequence of SEQ ID NO:19. In some embodiments, each of the two VHdomains comprises: (i) a CDR-H1 comprising the amino acid sequence ofSEQ ID NO:17; (ii) a CDR-H2 comprising the amino acid sequence of SEQ IDNO:18; and (iii) a CDR-H3 comprising the amino acid sequence of SEQ IDNO:19. In some embodiments, one or both of the two VH domains comprisesthe amino acid sequence of SEQ ID NO:23, or an amino acid sequencehaving at least 90%, at least 95%, or at least 99% sequence identity toSEQ ID NO:23. In some embodiments, one or both of the two VH domainscomprises the amino acid sequence of SEQ ID NO:29, or an amino acidsequence having at least 90%, at least 95%, or at least 99% sequenceidentity to SEQ ID NO:29. In some embodiments, at least one of the twoVL domains comprises: (i) a CDR-L1 comprising the amino acid sequence ofSEQ ID NO:20; (ii) a CDR-L2 comprising the amino acid sequence of SEQ IDNO:21; and (iii) a CDR-L3 comprising the amino acid sequence of SEQ IDNO:22. In some embodiments, each of the two VL domains comprises: (i) aCDR-L1 comprising the amino acid sequence of SEQ ID NO:20; (ii) a CDR-L2comprising the amino acid sequence of SEQ ID NO:21; and (iii) a CDR-L3comprising the amino acid sequence of SEQ ID NO:22. In some embodiments,one or both of the two VL domains comprises the amino acid sequence ofSEQ ID NO:24, or an amino acid sequence having at least 90%, at least95%, or at least 99% sequence identity to SEQ ID NO:24. In someembodiments, one or both of the two VL domains comprises the amino acidsequence of SEQ ID NO:30, or an amino acid sequence having at least 90%,at least 95%, or at least 99% sequence identity to SEQ ID NO:30. In someembodiments, each of the two single-chain polypeptides comprises, fromN-terminus to C-terminus: (a) a first VL domain of the two VL domains;(b) a first linker sequence; (c) a first VH domain of the two VHdomains; (d) a second linker sequence; (e) a second VL domain of the twoVL domains; (f) a third linker sequence; (g) a second VH domain of thetwo VH domains; (h) a fourth linker sequence; and (i) the antibody Fcdomain. In some embodiments, the first, second and third linkersequences each comprise two or more repeats of the amino acid sequenceof SEQ ID NO:25. In some embodiments, the first and the third linkersequences have the same sequence and comprise two repeats of SEQ IDNO:25. In some embodiments, the second linker sequence comprises fiverepeats of SEQ ID NO:25. In some embodiments, the fourth linker sequencecomprises the amino acid sequence of SEQ ID NO:26. In some embodiments,each of the two single-chain polypeptides comprises the amino acidsequence of SEQ ID NO:1, or an amino acid sequence having at least 90%,at least 95%, or at least 99% sequence identity to SEQ ID NO:1. In someembodiments, each of the two single-chain polypeptides is encoded by apolynucleotide comprising the polynucleotide sequence of SEQ ID NO:2. Insome embodiments, each of the two single-chain polypeptides comprises,from N-terminus to C-terminus: (a) a first VH domain of the two VHdomains; (b) a first linker sequence; (c) a first VL domain of the twoVL domains; (d) a second linker sequence; (e) a second VL domain of thetwo VL domains; (f) a third linker sequence; (g) a second VH domain ofthe two VH domains; (h) a fourth linker sequence; and (i) the antibodyFc domain. In some embodiments, each of the two single-chainpolypeptides comprises, from N-terminus to C-terminus: (a) a first VLdomain of the two VL domains; (b) a first linker sequence; (c) a firstVH domain of the two VH domains; (d) a second linker sequence; (e) asecond VH domain of the two VH domains; (f) a third linker sequence; (g)a second VL domain of the two VL domains; (h) a fourth linker sequence;and (i) the antibody Fc domain. In some embodiments, the first, secondor third linker sequence comprises two or more repeats of the amino acidsequence of SEQ IN NO:25. In some embodiments, the first, second orthird linker sequence comprises the amino acid sequence of SEQ ID NO:33,34, 35, or 36. In some embodiments, the first and the third linkersequences have the same sequence comprising five repeats of SEQ IDNO:25. In some embodiments, the second linker sequence comprises theamino acid sequence of SEQ ID NO:27. In some embodiments, the fourthlinker sequence comprises the amino acid sequence of SEQ ID NO:26. Insome embodiments, each of the two single-chain polypeptides comprisesthe amino acid sequence of SEQ ID NO:3, or an amino acid sequence havingat least 90%, at least 95%, or at least 99% sequence identity to SEQ IDNO:3. In some embodiments, each of the two single-chain polypeptides isencoded by a polynucleotide comprising the polynucleotide sequence ofSEQ ID NO:4. In some embodiments, each of the two single-chainpolypeptides comprises the amino acid sequence of SEQ ID NO:5. In someembodiments, each of the two single-chain polypeptides is encoded by apolynucleotide comprising the polynucleotide sequence of SEQ ID NO:6.

In another aspect, provided herein is a tetravalent antibody thatspecifically binds to human PSGL-1, the tetravalent antibody comprisinga dimer of two monomers, wherein each monomer of the dimer comprises anantibody heavy chain and an antibody light chain; wherein the antibodylight chain comprises: (i) two light chain variable (VL) domains,wherein each of the two VL domains comprises a CDR-L1, a CDR-L2, and aCDR-L3, (ii) a first heavy chain variable (VH) domain, and (iii) a lightchain constant (CL) domain; wherein the antibody heavy chain comprises:(i) a second heavy chain variable (VH) domain, and (ii) a heavy chainconstant region comprising a first heavy chain constant region (CH1)domain, an antibody hinge region, an second heavy chain constant region(CH2) domain, and a third heavy chain constant region (CH3) domain;wherein the first and the second VH domains each comprise a CDR-H1, aCDR-H2, and a CDR-H3, wherein each of the two VL domains forms a VH-VLbinding unit with a corresponding VH domain of the first and the secondVH domains, and wherein each of the two VH-VL binding units is specificfor human PSGL-1. In some embodiments, at least one of the first and thesecond VH domains comprises: (i) a CDR-H1 comprising the amino acidsequence of SEQ ID NO:17; (ii) a CDR-H2 comprising the amino acidsequence of SEQ ID NO:18; and (iii) a CDR-H3 comprising the amino acidsequence of SEQ ID NO:19. In some embodiments, the first and the secondVH domains each comprise: (i) a CDR-H1 comprising the amino acidsequence of SEQ ID NO:17; (ii) a CDR-H2 comprising the amino acidsequence of SEQ ID NO:18; and (iii) a CDR-H3 comprising the amino acidsequence of SEQ ID NO:19. In some embodiments, the first and/or thesecond VH domains comprise the amino acid sequence of SEQ ID NO:23, oran amino acid sequence having at least 90%, at least 95%, or at least99% sequence identity to SEQ ID NO:23. In some embodiments, the firstand/or the second VH domains comprise the amino acid sequence of SEQ IDNO:29, or an amino acid sequence having at least 90%, at least 95%, orat least 99% sequence identity to SEQ ID NO:29. In some embodiments, atleast one of the first and the second VL domains comprises: (i) a CDR-L1comprising the amino acid sequence of SEQ ID NO:20; (ii) a CDR-L2comprising the amino acid sequence of SEQ ID NO:21; and (iii) a CDR-L3comprising the amino acid sequence of SEQ ID NO:22. In some embodiments,the first and the second VL domains each comprise: (i) a CDR-L1comprising the amino acid sequence of SEQ ID NO:20; (ii) a CDR-L2comprising the amino acid sequence of SEQ ID NO:21; and (iii) a CDR-L3comprising the amino acid sequence of SEQ ID NO:22. In some embodiments,the first and/or the second VL domains comprise the amino acid sequenceof SEQ ID NO:24, or an amino acid sequence having at least 90%, at least95%, or at least 99% sequence identity to SEQ ID NO:24. In someembodiments, the first and/or the second VL domains comprise the aminoacid sequence of SEQ ID NO:30, or an amino acid sequence having at least90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:30. Insome embodiments, the antibody light chain comprises, from N-terminus toC-terminus: (a) the first VH domain; (b) a first linker sequence; (c) afirst VL domain of the two or more VL domains; (d) a second linkersequence; (e) a second VL domain of the two or more VL domains; and (f)the CL domain. In some embodiments, the CL domain is a kappa CL domain.In some embodiments, the first linker sequence comprises five repeats ofSEQ ID NO:25. In some embodiments, the second linker sequence comprisesthe amino acid sequence of SEQ ID NO:28. In some embodiments, theantibody light chain comprises the amino acid sequence of SEQ ID NO:7,or an amino acid sequence having at least 90%, at least 95%, or at least99% sequence identity to SEQ ID NO:7. In some embodiments, the antibodylight chain is encoded by a polynucleotide comprising the polynucleotidesequence of SEQ ID NO:8. In some embodiments, the antibody light chaincomprises, from N-terminus to C-terminus: (a) a first VL domain of thetwo VL domains; (b) the CL domain; (c) a first linker sequence; (d) thefirst VH domain; (e) a second linker sequence; and (f) a second VLdomain of the two VL domains. In some embodiments, the CL domain is akappa CL domain. In some embodiments, the first linker sequencecomprises two repeats of SEQ ID NO:25. In some embodiments, the secondlinker sequence comprises five repeats of SEQ ID NO:25. In someembodiments, the antibody light chain comprises the amino acid sequenceof SEQ ID NO:9. In some embodiments, the antibody light chain is encodedby a polynucleotide comprising the polynucleotide sequence of SEQ IDNO:10. In some embodiments, the antibody heavy chain comprises, fromN-terminus to C-terminus: (a) the second VH domain; and (b) a heavychain constant region comprising a first heavy chain constant region(CH1) domain, an antibody hinge region, an second heavy chain constantregion (CH2) domain, and a third heavy chain constant region (CH3)domain. In some embodiments, the antibody heavy chain comprises theamino acid sequence of SEQ ID NO:11, or an amino acid sequence having atleast 90%, at least 95%, or at least 99% sequence identity to SEQ IDNO:11. In some embodiments, the antibody heavy chain is encoded by apolynucleotide comprising the polynucleotide sequence of SEQ ID NO:12.

In another aspect, provided herein is a tetravalent antibody thatspecifically binds to human PSGL-1, the tetravalent antibody comprisinga dimer of two monomers, wherein each monomer of the dimer comprises anantibody heavy chain and an antibody light chain; wherein the antibodylight chain comprises: (i) a first heavy chain variable (VH) domain,(ii) a first light chain variable (VL) domain, and (iii) a light chainconstant (CL) domain; wherein the antibody heavy chain comprises: (i) asecond heavy chain variable (VH) domain, (ii) a second light chainvariable (VL) domain, and (iii) a heavy chain constant domain comprisinga first heavy chain constant region (CH1) domain, an antibody hingeregion, an second heavy chain constant region (CH2) domain, and a thirdheavy chain constant region (CH3) domain; wherein each of the first andsecond VL domains comprises a CDR-L1, a CDR-L2, and a CDR-L3; whereineach of the first and second VH domains comprises a CDR-H1, a CDR-H2,and a CDR-H3; wherein each of the first and second VL domains forms aVH-VL binding unit with a corresponding VH domain of the first andsecond VH domains; and wherein each of the two VH-VL binding units isspecific for human PSGL-1. In some embodiments, at least one of thefirst and second VH domains comprises: (i) a CDR-H1 comprising the aminoacid sequence of SEQ ID NO:17; (ii) a CDR-H2 comprising the amino acidsequence of SEQ ID NO:18; and (iii) a CDR-H3 comprising the amino acidsequence of SEQ ID NO:19. In some embodiments, the first and the secondVH domains each comprise: (i) a CDR-H1 comprising the amino acidsequence of SEQ ID NO:17; (ii) a CDR-H2 comprising the amino acidsequence of SEQ ID NO:18; and (iii) a CDR-H3 comprising the amino acidsequence of SEQ ID NO:19. In some embodiments, the first and/or thesecond VH domains comprise the amino acid sequence of SEQ ID NO:23. Insome embodiments, the first and/or the second VH domains comprise theamino acid sequence of SEQ ID NO:29. In some embodiments, at least oneof the first and second VL domains comprises: (i) a CDR-L1 comprisingthe amino acid sequence of SEQ ID NO:20; (ii) a CDR-L2 comprising theamino acid sequence of SEQ ID NO:21; and (iii) a CDR-L3 comprising theamino acid sequence of SEQ ID NO:22. In some embodiments, the first andthe second VL domains each comprise: (i) a CDR-L1 comprising the aminoacid sequence of SEQ ID NO:20; (ii) a CDR-L2 comprising the amino acidsequence of SEQ ID NO:21; and (iii) a CDR-L3 comprising the amino acidsequence of SEQ ID NO:22. In some embodiments, the first and/or thesecond VL domains comprise the amino acid sequence of SEQ ID NO:24. Insome embodiments, the first and/or the second VL domains comprise theamino acid sequence of SEQ ID NO:30. In some embodiments, the antibodylight chain comprises, from N-terminus to C-terminus: (a) the first VHdomain; (b) a first linker sequence; (c) the first VL domain; and (d)the CL domain. In some embodiments, the CL domain is a kappa CL domain.In some embodiments, the first linker sequence comprises five repeats ofSEQ ID NO:25. In some embodiments, the antibody light chain comprisesthe amino acid sequence of SEQ ID NO:13. In some embodiments, theantibody light chain is encoded by a polynucleotide comprising thepolynucleotide sequence of SEQ ID NO:14. In some embodiments, theantibody heavy chain comprises, from N-terminus to C-terminus: (a) thesecond VH domain; (b) a second linker sequence; (c) the second VLdomain; and (d) the heavy chain constant region comprising the firstheavy chain constant region (CH1) domain, the antibody hinge region, thesecond heavy chain constant region (CH2) domain, and the third heavychain constant region (CH3) domain. In some embodiments, the secondlinker sequence comprises five repeats of SEQ ID NO:25. In someembodiments, the antibody heavy chain comprises the amino acid sequenceof SEQ ID NO:15. In some embodiments, the antibody heavy chain isencoded by a polynucleotide comprising the polynucleotide sequence ofSEQ ID NO:16.

In some embodiments of any of the above embodiments, the antibody Fcdomain is a human antibody Fc domain. In some embodiments, the antibodyFc domain is a human IgG4 Fc domain. In some embodiments, the human IgG4Fc domain comprises a hinge region sequence comprising one or more aminoacid substitutions that result in reduced IgG4 shuffling, as compared toan IgG4 hinge region lacking the one or more amino acid substitutions.In some embodiments, the human IgG4 Fc domain comprises a hinge regionsequence comprising a serine to proline substitution at amino acid 228,numbering according to EU index. In some embodiments of any of the aboveembodiments, the antibody hinge region comprises a serine to prolinesubstitution at amino acid 228, numbering according to EU index. In someembodiments, a tetravalent antibody of the present disclosure displaysenhanced induction of apoptosis in a target cell (e.g., a cellexpressing human PSGL-1 or an epitope thereof) as compared to aconventional (e.g., bivalent) antibody having one or more VH or VLdomains in common with the tetravalent antibody. In some embodiments, atetravalent antibody of the present disclosure displays enhancedinhibition of DTH (e.g., in a trans vivo animal model) as compared to aconventional (e.g., bivalent) antibody having one or more VH or VLdomains in common with the tetravalent antibody.

In another aspect, provided herein is an isolated polynucleotideencoding the tetravalent antibody of any one of the above embodiments.In some embodiments, the isolated polynucleotide comprises apolynucleotide sequence selected from the group consisting of SEQ IDNOs:2, 4, 6, 8, 10, 12, 14, and 16. In another aspect, provided hereinis a vector comprising the isolated polynucleotide of any of the aboveembodiments. In another aspect, provided herein is a host cellcomprising the polynucleotide of any of the above embodiments and/or thevector of any of the above embodiments. In another aspect, providedherein is a method of producing a tetravalent antibody comprisingculturing the host cell of any of the above embodiments so that thetetravalent antibody is produced. In some embodiments, the methodfurther comprises recovering the tetravalent antibody from the hostcell.

In another aspect, provided herein is a pharmaceutical compositioncomprising the tetravalent antibody of any one of the above embodimentsand a pharmaceutically acceptable carrier. In another aspect, providedherein is a kit comprising the tetravalent antibody of any one of theabove embodiments and an optional pharmaceutically acceptable carrier.In some embodiments, the kit further comprises a package insertcomprising instructions for administration of the tetravalent antibodyto treat a T-cell mediated inflammatory disease or condition. In someembodiments, the kit further comprises a package insert comprisinginstructions for administration of the tetravalent antibody before,concurrently with, and/or after a transfusion or transplantation. Inanother aspect, provided herein is the tetravalent antibody of any oneof the above embodiments for use in treating a T-cell mediatedinflammatory disease or condition. In another aspect, provided herein isthe tetravalent antibody of any one of the above embodiments for use intreating an individual in need of a transfusion or transplantation. Inanother aspect, provided herein is a use of the tetravalent antibody ofany one of the above embodiments in the manufacture of a medicament fortreating a T-cell mediated inflammatory disease or condition. In anotheraspect, provided herein is a use of the tetravalent antibody of any oneof the above embodiments in the manufacture of a medicament for treatingan individual in need of a transfusion or transplantation. In anotheraspect, provided herein is a method of treating a T-cell mediatedinflammatory disease or condition, the method comprising administeringto a subject in need thereof a therapeutically effective amount of thetetravalent antibody of any one of the above embodiments. In anotheraspect, provided herein is a method for treating an individual in needof a transfusion or transplantation, comprising administering to theindividual a therapeutically effective amount of the tetravalentantibody of any one of the above embodiments before, concurrently with,and/or after the transfusion or transplantation. In some embodiments,the T-cell mediated inflammatory disease is an autoimmune disease. Insome embodiments, the T-cell mediated inflammatory disease is selectedfrom the group consisting of psoriasis, psoriatic arthritis, rheumatoidarthritis, Crohn's disease, ankylosing spondylitis, type I diabetes,ulcerative colitis, multiple sclerosis, and graft versus host disease(GVHD). In some embodiments, the psoriasis is plaque psoriasis, chronicplaque psoriasis, guttate psoriasis, inverse psoriasis, pustularpsoriasis, or erythrodermic psoriasis. In some embodiments, thetransplantation is a transplantation of a tissue selected from the groupconsisting of bone marrow, kidney, heart, liver, neuronal tissue, lung,pancreas, skin, and intestine. In some embodiments, the transfusion is atransfusion comprising one or more of white blood cells, red bloodcells, and platelets.

It is to be understood that one, some, or all of the properties of thevarious embodiments described herein may be combined to form otherembodiments of the present invention. These and other aspects of theinvention will become apparent to one of skill in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A & 1B provide schematics illustrating exemplary tetravalentantibodies in accordance with some embodiments. FIG. 1A illustrates thefollowing exemplary formats: (1) a dimer composed of two single-chaindiabodies fused to an Fc domain (scDb₂-Fc), showing linker sequences:(GGGGS)₅ (SEQ ID NO:33), GGGGSAAA (SEQ ID NO:26) and (GGGGS)₂ (SEQ IDNO:34)/(GGGGS)₂G (SEQ ID NO:35)/(GGGGS)₂GG (SEQ ID NO:36); (2) twodifferent formats, each having a dimer of two tandem single-chainvariable fragment units (taFv₂-Fc), showing identical linker sequencesfor both formats: (GGGGS)₅ (SEQ ID NO:33), ASTGS (SEQ ID NO:27),GGGGSAAA (SEQ ID NO:26); and (3) three different formats based onsingle-chain variable fragments (scFv-IgG), showing: scFv₂-LC-IgG4plinker sequences (GGGGS)₅ (SEQ ID NO:33) and ASTGSG₄S (SEQ ID NO:28),LC-scFv₂-IgG4p linker sequences (GGGGS)₂ (SEQ ID NO:34) and (GGGGS)₅(SEQ ID NO:33), scFv₄-crlG4p linker sequences (GGGGS)₅ (SEQ ID NO:33).FIG. 1B provides another illustration of the three scFv-based formats,with the variable fragments shaded and V2 scFvs indicated.

FIGS. 2A-2C show the verification of the molecular weights and basicstructures of exemplary tetravalent antibodies by SDS-PAGE followed byCoomassie blue staining. Non-reducing (FIGS. 2A & 2B) and reducing (FIG.2C) conditions are shown.

DETAILED DESCRIPTION

Provided herein are tetravalent antibodies that specifically bind tohuman PSGL-1. The present disclosure is based at least in part on thefinding described herein that certain tetravalent anti-PSGL-1 antibodiesshow enhanced efficacy compared to the parental anti-PSGL-1 antibodyboth in vitro and trans vivo. These tetravalent antibodies displayedhigher potency for apoptosis induction and enhanced efficacy in a transvivo model for delayed type hypersensitivity (DTH) than the parentalanti-PSGL-1 antibody. Further provided herein are isolatedpolynucleotides, vectors, host cells, pharmaceutical compositions, kits,uses, and methods related to the tetravalent antibodies. For example,the tetravalent antibodies of the present disclosure may find use intreating a T-cell mediated inflammatory disease, or administrationbefore, concurrently with, and/or after a transfusion ortransplantation.

In some embodiments, the tetravalent antibodies of the presentdisclosure comprise a dimer of two monomers, wherein each monomer of thedimer comprises a single-chain polypeptide comprising: (a) two lightchain variable (VL) domains, wherein each of the two VL domainscomprises a CDR-L1, a CDR-L2, and a CDR-L3; (b) two heavy chain variable(VH) domains, wherein each of the two VH domains comprises a CDR-H1, aCDR-H2, and a CDR-H3; and (c) an antibody Fc domain, wherein each of thetwo VL domains forms a VH-VL binding unit with a corresponding VH domainof the two VH domains, and wherein each of the two VH-VL binding unitsis specific for human PSGL-1. In other embodiments, the tetravalentantibodies of the present disclosure comprise a dimer of two monomers,wherein each monomer of the dimer comprises an antibody heavy chain andan antibody light chain; wherein the antibody light chain comprises: (i)two light chain variable (VL) domains, wherein each of the two VLdomains comprises a CDR-L1, a CDR-L2, and a CDR-L3, (ii) a first heavychain variable (VH) domain, and (iii) a light chain constant (CL)domain; wherein the antibody heavy chain comprises: (i) a second heavychain variable (VH) domain, and (ii) a heavy chain constant regioncomprising a first heavy chain constant region (CH1) domain, an antibodyhinge region, an second heavy chain constant region (CH2) domain, and athird heavy chain constant region (CH3) domain; wherein the first andthe second VH domains each comprise a CDR-H1, a CDR-H2, and a CDR-H3,wherein each of the two VL domains forms a VH-VL binding unit with acorresponding VH domain of the first and the second VH domains, andwherein each of the two VH-VL binding units is specific for humanPSGL-1. In other embodiments, the tetravalent antibodies of the presentdisclosure comprise a dimer of two monomers, wherein each monomer of thedimer comprises an antibody heavy chain and an antibody light chain;wherein the antibody light chain comprises: (i) a first heavy chainvariable (VH) domain, (ii) a first light chain variable (VL) domain, and(iii) a light chain constant (CL) domain; wherein the antibody heavychain comprises: (i) a second heavy chain variable (VH) domain, (ii) asecond light chain variable (VL) domain, and (iii) a heavy chainconstant region comprising a first heavy chain constant region (CH1)domain, an antibody hinge region, an second heavy chain constant region(CH2) domain, and a third heavy chain constant region (CH3) domain;wherein each of the first and second VL domains comprises a CDR-L1, aCDR-L2, and a CDR-L3; wherein each of the first and second VH domainscomprises a CDR-H1, a CDR-H2, and a CDR-H3; wherein each of the firstand second VL domains forms a VH-VL binding unit with a corresponding VHdomain of the first and second VH domains; and wherein each of the twoVH-VL binding units is specific for human PSGL-1.

I. Definitions

An “antibody” is an immunoglobulin molecule capable of specific bindingto a target, such as a carbohydrate, polynucleotide, lipid, polypeptide,etc., through at least one antigen recognition site, located in thevariable region of the immunoglobulin molecule. As used herein, the termencompasses not only intact polyclonal or monoclonal antibodies, butalso polypeptides comprising fragments thereof (such as Fab, Fab′,F(ab′)₂, Fv); single-chain variable fragments (scFv), single-chaindiabodies (scDbs), tandem single-chain variable fragment (scFv) units(termed taFv for tandem scFv), and mutants or other configurationsthereof; fusion proteins comprising an antibody portion; and any othermodified configuration of the immunoglobulin molecule that comprises anantigen recognition site.

As used herein, a “tetravalent” antibody may refer to an antibody thatcomprises four antibody VH-VL binding units, with each VH-VL bindingunit comprising an antibody VH domain and an antibody VL domain. As usedherein, references to a “monomer” of a tetravalent antibody may includeboth single-chain polypeptides and multiple-chain polypeptides. Forexample, a monomer may refer to a single-chain polypeptide, or it mayrefer to an antibody heavy chain-light chain unit, where the heavy chainand light chain are encoded by separate polynucleotides and/or areformed from the association of separate polypeptides.

An antibody includes an antibody of any class, such as IgG, IgA, or IgM(or sub-class thereof), and the antibody need not be of any particularclass. Depending on the antibody amino acid sequence of the constantdomain of its heavy chains, immunoglobulins can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Theheavy chain constant domains that correspond to the different classes ofimmunoglobulins are called alpha, delta, epsilon, gamma, and mu,respectively. The subunit structures and three-dimensionalconfigurations of different classes of immunoglobulins are well known.

The antibodies of the present disclosure are further intended to includebispecific, multispecific, chimeric, humanized, and recombinantlyconstructed molecules having affinity for a polypeptide conferred by atleast one CDR region of the antibody. Single domain antibodies which areeither the variable domain of an antibody heavy chain or the variabledomain of an antibody light chain are known in the art. See, e.g., Holtet al., Trends Biotechnol. 21:484-490, 2003. Methods of makingantibodies comprising either the variable domain of an antibody heavychain or the variable domain of an antibody light chain, containingthree of the six naturally occurring complementarity determining regionsfrom an antibody, are also known in the art. See, e.g., Muyldermans,Rev. Mol. Biotechnol. 74:277-302, 2001.

As used herein, “monoclonal antibody” refers to an antibody ofsubstantially homogeneous antibodies, i.e., the individual antibodiescomprising the population are identical except for possiblenaturally-occurring mutations that may be present in minor amounts.Monoclonal antibodies are generally highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations, which typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen. Themodifier “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies, andis not to be construed as requiring production of the antibody by anyparticular method. For example, the monoclonal antibodies to be used inaccordance with the present disclosure may be made by the hybridomamethod first described by Kohler and Milstein, 1975, Nature, 256:495, ormay be made by recombinant DNA methods such as described in U.S. Pat.No. 4,816,567. The monoclonal antibodies may also be isolated from phagelibraries generated using the techniques described in McCafferty et al.,1990, Nature, 348:552-554, for example.

As used herein, a “chimeric antibody” refers to an antibody having avariable region or part of a variable region from a first species and aconstant region from a second species. An intact chimeric antibodycomprises two copies of a chimeric light chain and two copies of achimeric heavy chain. The production of chimeric antibodies is known inthe art (Cabilly et al. (1984), Proc. Natl. Acad. Sci. USA,81:3273-3277; Harlow and Lane (1988), Antibodies: a Laboratory Manual,Cold Spring Harbor Laboratory). Typically, in these chimeric antibodies,the variable region of both light and heavy chains mimics the variableregions of antibodies derived from one species of mammal, while theconstant portions are homologous to the sequences in antibodies derivedfrom another. One clear advantage to such chimeric forms is that, forexample, the variable regions can conveniently be derived from presentlyknown sources using readily available hybridomas or B-cells fromnon-human host organisms in combination with constant regions derivedfrom, for example, human cell preparations. While the variable regionhas the advantage of ease of preparation, and the specificity is notaffected by its source, the constant region being human is less likelyto elicit an immune response from a human subject when the antibodiesare injected than would the constant region from a non-human source.However, the definition is not limited to this particular example. Insome embodiments, amino acid modifications are made in the variableand/or constant region.

As used herein, “humanized” antibodies refer to forms of non-human(e.g., murine) antibodies that are specific chimeric immunoglobulins,immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab′,F(ab′)₂, or other antigen-binding subsequences of antibodies) thatcontain minimal sequence derived from non-human immunoglobulin. For themost part, humanized antibodies are human immunoglobulins (recipientantibody) in which residues from a complementary determining region(CDR) of the recipient are replaced by residues from a CDR of anon-human species (donor antibody) such as mouse, rat, or rabbit havingthe desired specificity, affinity, and capacity. In some instances, Fvframework region (FR) residues of the human immunoglobulin are replacedby corresponding non-human residues. Furthermore, the humanized antibodymay comprise residues that are found neither in the recipient antibodynor in the imported CDR or framework sequences, but are included tofurther refine and optimize antibody performance. In general, thehumanized antibody will comprise substantially all of at least one, andtypically two, variable domains in which all or substantially all of theCDR regions correspond to those of a non-human immunoglobulin and all orsubstantially all of the FR regions are those of a human immunoglobulinconsensus sequence. The humanized antibody optimally also will compriseat least a portion of an immunoglobulin constant region or domain (e.g.,an Fc domain), typically that of a human immunoglobulin. Antibodies mayhave Fc regions modified as described in WO 99/58572. Other forms ofhumanized antibodies have one or more CDRs (one, two, three, four, five,or six) which are altered with respect to the original antibody, whichare also termed one or more CDRs “derived from” one or more CDRs fromthe original antibody.

As used herein, “human antibody” means an antibody having an amino acidsequence corresponding to that of an antibody produced by a human and/orhas been made using any of the techniques for making human antibodiesknown in the art or disclosed herein. This definition of a humanantibody includes antibodies comprising at least one human heavy chainpolypeptide or at least one human light chain polypeptide. One suchexample is an antibody comprising murine light chain and human heavychain polypeptides. Human antibodies can be produced using varioustechniques known in the art. In one embodiment, the human antibody isselected from a phage library, where that phage library expresses humanantibodies (Vaughan et al., 1996, Nature Biotechnology, 14:309-314;Sheets et al., 1998, PNAS, (USA) 95:6157-6162; Hoogenboom and Winter,1991, J. Mol. Biol., 227:381; Marks et al., 1991, J. Mol. Biol.,222:581). Human antibodies can also be made by introducing humanimmunoglobulin loci into transgenic animals, e.g., mice in which theendogenous immunoglobulin genes have been partially or completelyinactivated. This approach is described in U.S. Pat. Nos. 5,545,807;5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016.Alternatively, the human antibody may be prepared by immortalizing humanB-lymphocytes that produce an antibody directed against a target antigen(such B-lymphocytes may be recovered from an individual or may have beenimmunized in vitro). See, e.g., Cole et al., Monoclonal Antibodies andCancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., 1991, J.Immunol., 147 (1):86-95; and U.S. Pat. No. 5,750,373.

A “variable region” (the term “variable domain” may be usedinterchangeably herein) of an antibody refers to the variable region ofthe antibody light chain (VL) or the variable region of the antibodyheavy chain (VH), either alone or in combination. The variable regionsof the heavy and light chain (VH and VL domains, respectively) eachconsist of four framework regions (FR) connected by threecomplementarity determining regions (CDRs) also known as hypervariableregions. The CDRs in each chain are held together in close proximity bythe FRs and, with the CDRs from the other chain, contribute to theformation of the antigen-binding site of antibodies. There are at leasttwo techniques for determining CDRs: (1) an approach based oncross-species sequence variability (i.e., Kabat et al. Sequences ofProteins of Immunological Interest, (5th ed., 1991, National Institutesof Health, Bethesda Md.)); and (2) an approach based on crystallographicstudies of antigen-antibody complexes (Al-lazikani et al (1997) J.Molec. Biol. 273:927-948)). As used herein, a CDR may refer to CDRsdefined by either approach or by a combination of both approaches.

A number of HVR delineations are in use and are encompassed herein. TheKabat Complementarity Determining Regions (CDRs) are based on sequencevariability and are the most commonly used (Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). Chothia refersinstead to the location of the structural loops (Chothia and Lesk, J.Mol. Biol. 196:901-917 (1987)). The AbM HVRs represent a compromisebetween the Kabat HVRs and Chothia structural loops, and are used byOxford Molecular's AbM antibody modeling software. The “contact” HVRsare based on an analysis of the available complex crystal structures.The residues from each of these HVRs are noted below.

Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1H31-H35B H26-H35B H26-H32 H30-H35B (Kabat numbering) H1 H31-H35 H26-H35H26-H32 H30-H35 (Chothia numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58H3 H95-H102 H95-H102 H96-H101 H93-H101

The Kabat numbering system is generally used when referring to a residuein the variable domain (approximately residues 1-107 of the light chainand residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences ofImmunological Interest. 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)). The “EU numbering system”or “EU index” is generally used when referring to a residue in animmunoglobulin heavy chain constant region (e.g., the EU index reportedin Kabat et al., supra, or and Edelman, G. M. et al. (1969) Proc. Natl.Acad. Sci. USA 63:78-85).

“Fv” as used herein may refer to the minimum antibody fragment whichcontains a complete antigen-recognition and -binding site. This fragmenttypically consists of a dimer of one heavy- and one light-chain variableregion domain in tight, non-covalent association. From the folding ofthese two domains emanate six hypervariable loops (3 loops each from theH and L chain) that contribute the amino acid residues for antigenbinding and confer antigen binding specificity to the antibody. However,even a single variable domain (or half of an Fv comprising only threeHVRs specific for an antigen) has the ability to recognize and bindantigen, although at a lower affinity than the entire binding site.

A “constant region” (the term “constant domain” may be usedinterchangeably herein) of an antibody refers to the constant region ofthe antibody light chain (CL) or the constant region of the antibodyheavy chain (CH), either alone or in combination. A constant region ofan antibody generally provides structural stability and other biologicalfunctions such as antibody chain association, secretion, transplacentalmobility, and complement binding, but is not involved with binding tothe antigen. The amino acid sequence and corresponding exon sequences inthe genes of the constant region is dependent upon the species fromwhich it is derived; however, variations in the amino acid sequenceleading to allotypes is relatively limited for particular constantregions within a species. The variable region of each chain is joined tothe constant region by a linking polypeptide sequence. The linkagesequence is coded by a “J” sequence in the light chain gene, and acombination of a “D” sequence and a “J” sequence in the heavy chaingene. Depending on the antibody isotype, a heavy chain constant regionmay include a CH1 domain, a hinge region, a CH2 domain, a CH3 domain,and/or a CH4 domain. In certain embodiments, a heavy chain constantregion comprises a CH1 domain, a hinge region, a CH2 domain, and a CH3domain.

The term “Fc region” (the term “Fc domain” may be used interchangeablyherein) herein is used to define a C-terminal region of animmunoglobulin heavy chain, including native-sequence Fc regions andvariant Fc regions. The boundaries of the Fc region of an immunoglobulinheavy chain might vary; in some embodiments, the Fc region may includeone or more amino acids of the hinge region. In some embodiments, thehuman IgG heavy-chain Fc region is defined to stretch from an amino acidresidue at EU position 216 to the carboxyl-terminus thereof. Suitablenative-sequence Fc regions for use in the antibodies of the presentdisclosure include human IgG1, IgG2 (IgG2A, IgG2B), IgG3 and IgG4.

“Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibodyfragments that comprise the V_(H) and V_(L) antibody domains connectedinto a single polypeptide chain. Preferably, the sFv polypeptide furthercomprises a polypeptide linker between the V_(H) and VL domains whichenables the sFv to form the desired structure for antigen binding. For areview of the sFv, see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994).

The term “diabodies” refers to antibody fragments prepared byconstructing sFv fragments (see preceding paragraph) with short linkers(e.g., about 5-12 residues) between the VH and V_(L) domains such thatinter-chain but not intra-chain pairing of the V domains is achieved,thereby resulting in a bivalent fragment, i.e., a fragment having twoantigen-binding sites. Bispecific diabodies are heterodimers of two“crossover” sFv fragments in which the V_(H) and V_(L) domains of thetwo antibodies are present on different polypeptide chains. Diabodiesare described in greater detail in, for example, EP 404,097; WO93/11161; Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448(1993).

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.

As used herein, “antibody-dependent cell-mediated cytotoxicity” and“ADCC” refer to a cell-mediated reaction in which nonspecific cytotoxiccells that express Fc receptors (FcRs) (e.g., natural killer (NK) cells,neutrophils, or macrophages) recognize bound antibody on a target celland subsequently cause lysis of the target cell. ADCC activity of amolecule of interest can be assessed using an in vitro ADCC assay, suchas that described in U.S. Pat. No. 5,500,362 or 5,821,337. Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and NK cells. Alternatively, or additionally, ADCC activityof the molecule of interest may be assessed in vivo, e.g., in a animalmodel such as that disclosed in Clynes et al., 1998, PNAS (USA),95:652-656.

“Complement dependent cytotoxicity” and “CDC” refer to the lysing of atarget in the presence of complement. The complement activation pathwayis initiated by the binding of the first component of the complementsystem (C1q) to a molecule (e.g., an antibody) complexed with a cognateantigen. To assess complement activation, a CDC assay, e.g., asdescribed in Gazzano-Santoro et al., J. Immunol. Methods, 202:163(1996), may be performed.

The terms “polypeptide,” “oligopeptide,” “peptide,” and “protein” areused interchangeably herein to refer to polymers of amino acids of anylength. The polymer may be linear or branched, it may comprise modifiedamino acids, and it may be interrupted by non-amino acids. The termsalso encompass an amino acid polymer that has been modified naturally orby intervention; for example, disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling component. Alsoincluded within the definition are, for example, polypeptides containingone or more analogs of an amino acid (including, for example, unnaturalamino acids, etc.), as well as other modifications known in the art. Itis understood that, because the polypeptides of the present disclosureare based upon a tetravalent antibody, the polypeptides can occur assingle chains or associated chains.

“Polynucleotide,” or “nucleic acid,” as used interchangeably herein,refer to polymers of nucleotides of any length, and include DNA and/orRNA. The nucleotides can be deoxyribonucleotides, ribonucleotides,modified nucleotides or bases, and/or their analogs, or any substratethat can be incorporated into a polymer by DNA or RNA polymerase. Apolynucleotide may comprise modified nucleotides, such as methylatednucleotides and their analogs. If present, modification to thenucleotide structure may be imparted before or after assembly of thepolymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter polymerization, such as by conjugation with a labeling component.Other types of modifications include, for example, “caps,” substitutionof one or more of the naturally occurring nucleotides with an analog,internucleotide modifications such as, for example, those with unchargedlinkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates,cabamates, etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example, proteins (e.g., nucleases, toxins, antibodies, signalpeptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine,psoralen, etc.), those containing chelators (e.g., metals, radioactivemetals, boron, oxidative metals, etc.), those containing alkylators,those with modified linkages (e.g., alpha anomeric nucleic acids, etc.),as well as unmodified forms of the polynucleotide(s). Further, any ofthe hydroxyl groups ordinarily present in the sugars may be replaced,for example, by phosphonate groups, phosphate groups, protected bystandard protecting groups, or activated to prepare additional linkagesto additional nucleotides, or may be conjugated to solid supports. The5′ and 3′ terminal OH can be phosphorylated or substituted with aminesor organic capping group moieties of from 1 to 20 carbon atoms. Otherhydroxyls may also be derivatized to standard protecting groups.Polynucleotides can also contain analogous forms of ribose ordeoxyribose sugars that are generally known in the art, including, forexample, 2′-O-methyl-, 2′-O-allyl, 2′-fluoro- or 2′-azido-ribose,carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such asarabinose, xyloses, lyxoses, pyranose sugars, furanose sugars,sedoheptuloses, acyclic analogs, and abasic nucleoside analogs such asmethyl ribosides. One or more phosphodiester linkages may be replaced byalternative linking groups. These alternative linking groups include,but are not limited to, embodiments wherein phosphate is replaced byP(O)S (“thioate”), P(S)S (“dithioate”), “(O)NR₂ (“amidate”), P(O)R,P(O)OR′, CO, or CH₂ (“formacetal”), in which each R or R′ isindependently H or substituted or unsubstituted alkyl (1-20 C)optionally containing an ether (—O—) linkage, aryl, alkenyl, cycloalkyl,cycloalkenyl, or araldyl. Not all linkages in a polynucleotide need beidentical. The preceding description applies to all polynucleotidesreferred to herein, including RNA and DNA.

As used herein, “vector” means a construct that is capable of deliveringand desirably expressing one or more gene(s) or sequence(s) of interestin a host cell. Examples of vectors include, but are not limited to,viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid orphage vectors, DNA or RNA expression vectors associated with cationiccondensing agents, DNA or RNA expression vectors encapsulated inliposomes, and certain eukaryotic cells, such as producer cells.

As used herein, “expression control sequence” means a nucleic acidsequence that directs transcription of a nucleic acid. An expressioncontrol sequence can be a promoter, such as a constitutive or aninducible promoter, or an enhancer. The expression control sequence isoperably linked to the nucleic acid sequence to be transcribed.

As used herein, an “effective dosage” or “therapeutically effectiveamount” of drug, compound, or pharmaceutical composition is an amountsufficient to effect beneficial, desired, and/or therapeutic results.For prophylactic use, beneficial or desired results include results suchas eliminating or reducing the risk, lessening the severity, or delayingthe onset of the disease, including biochemical, histological and/orbehavioral symptoms of the disease, its complications and intermediatepathological phenotypes presenting during development of the disease.For therapeutic use, beneficial or desired results include clinicalresults such as decreasing one or more symptoms resulting from thedisease, increasing the quality of life of those suffering from thedisease, decreasing the dose of other medications required to treat thedisease, enhancing effect of another medication such as via targeting,delaying the progression of the disease, and/or prolonging survival. Inthe case of treating an individual awaiting a transplantation, forexample, an effective amount of the drug may reduce to some extent thelevel of alloantibodies and/or PRA in the individual. In the case oftreating an individual receiving a transplantation or transfusion, aneffective amount of the drug may have the effect in and/or relieving tosome extent one or more of the symptoms or conditions (such as graftrejection) associated with the transplantation or transfusion. Aneffective amount can be administered in one or more administrations. Forpurposes of the present disclosure, an effective amount of drug,compound, or pharmaceutical composition is an amount sufficient toaccomplish prophylactic or therapeutic treatment either directly orindirectly. An effective dosage can be administered in one or moreadministrations. For purposes of the present disclosure, an effectivedosage of drug, compound, or pharmaceutical composition is an amountsufficient to accomplish prophylactic or therapeutic treatment eitherdirectly or indirectly. As is understood in the clinical context, aneffective dosage of a drug, compound, or pharmaceutical composition mayor may not be achieved in conjunction with another drug, compound, orpharmaceutical composition. Thus, an “effective dosage” may beconsidered in the context of administering one or more therapeuticagents, and a single agent may be considered to be given in an effectiveamount if, in conjunction with one or more other agents, a desirableresult may be or is achieved.

As used herein, “in conjunction with” refers to administration of onetreatment modality in addition to another treatment modality. As such,“in conjunction with” refers to administration of one treatment modalitybefore, during, or after administration of the other treatment modalityto the individual.

As used herein, “treatment” or “treating” is an approach for obtainingbeneficial or desired results, including desirably clinical results.Beneficial, desired, and/or therapeutic clinical results include, butare not limited to, one or more of the following: reducing or abrogatingone or more symptoms of inflammation or autoimmunity (e.g., stemmingfrom a T-cell mediated inflammatory disease), increasing the likelihoodof a successful patient outcome and/or mitigating one or morecontraindications or detrimental outcomes related to a medical treatment(e.g., related to a transplantation or transfusion), decreasing symptomsresulting from the disease, increasing the quality of life of thosesuffering from the disease, decreasing the dose of other medicationsrequired to treat the disease, delaying the progression of the disease,and/or prolonging survival of individuals.

As used herein, “delaying development of a disease” means to defer,hinder, slow, retard, stabilize, and/or postpone development of thedisease (such as cancer). This delay can be of varying lengths of time,depending on the history of the disease and/or individual being treated.As is evident to one skilled in the art, a sufficient or significantdelay can, in effect, encompass prevention, in that the individual doesnot develop the disease. For example, a symptom of an inflammatorydisease, such as a T-cell mediated inflammatory disease, may be delayed.

An “individual” or a “subject” is a mammal, more desirably a human.Mammals also include, but are not limited to, farm animals, sportanimals, pets (such as cats, dogs, or horses), primates, mice, and rats.

As used herein, the term “specifically recognizes” or “specificallybinds” refers to measurable and reproducible interactions such asattraction or binding between a target and an antibody (e.g., afull-length antibody, an antibody fragment, or an antibody VH-VL bindingunit) that is determinative of the presence of the target in thepresence of a heterogeneous population of molecules including biologicalmolecules. For example, an antibody, antibody fragment, or antibodyVH-VL binding unit that specifically or preferentially binds to anepitope is an antibody that binds this epitope with greater affinity,avidity, more readily, and/or with greater duration than it binds toother epitopes of the target or non-target epitopes. It is alsounderstood by reading this definition that, for example, an antibody,antibody fragment, or antibody VH-VL binding unit that specifically orpreferentially binds to a first target may or may not specifically orpreferentially bind to a second target. As such, “specific binding” or“preferential binding” does not necessarily require (although it caninclude) exclusive binding. An antibody, antibody fragment, or antibodyVH-VL binding unit that specifically binds to a target may have anassociation constant of greater than or about 10³ M⁻¹ or about 10⁴ M⁻¹,sometimes about 10⁵ M⁻¹ or about 10⁶ M⁻¹, in other instances about 10⁶M⁻¹ or about 10⁷ M⁻¹, about 10⁸ M⁻¹ to about 10⁹ M⁻¹, or about 10¹⁰ M⁻¹to about 10¹¹M⁻¹ or higher. A variety of immunoassay formats can be usedto select antibodies, antibody fragments, or antibody VH-VL bindingunits that are specifically immunoreactive with a particular protein.For example, solid-phase ELISA immunoassays are routinely used to selectmonoclonal antibodies specifically immunoreactive with a protein. See,e.g., Harlow and Lane (1988) Antibodies, A Laboratory Manual, ColdSpring Harbor Publications, New York, for a description of immunoassayformats and conditions that can be used to determine specificimmunoreactivity.

A “package insert” refers to instructions customarily included incommercial packages of medicaments that contain information about theindications customarily included in commercial packages of medicamentsthat contain information about the indications, usage, dosage,administration, contraindications, other medicaments to be combined withthe packaged product, and/or warnings concerning the use of suchmedicaments, etc.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly indicatesotherwise. For example, reference to an “antibody” is a reference tofrom one to many antibodies, such as molar amounts, and includesequivalents thereof known to those skilled in the art, and so forth.

Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X.”

It is understood that aspect and variations of the present disclosuredescribed herein include “consisting” and/or “consisting essentially of”aspects and variations.

II. Tetravalent Antibodies

Certain aspects of the present disclosure relate to tetravalentantibodies that specifically bind to human PSGL-1. In some embodiments,a tetravalent antibody of the present disclosure comprises a dimer oftwo monomers. As described infra, the monomers may be coupled using anymeans known in the art, including without limitation wild-typeinteractions between antibody Fc domains or regions, altered or mutatedinteractions between antibody Fc domains or regions (e.g., using a hingeregion mutation described herein), or other artificial covalent ornon-covalent interactions (e.g., cross-linking or a linker). Exemplarytetravalent antibodies and antibody formats are described below andillustrated in FIGS. 1A & 1B.

Human PSGL-1 may also be referred to as selectin P ligand, SELPG, CLA,CD162, or PSGL1. In some embodiments, a tetravalent antibody of thepresent disclosure binds to a polypeptide encoded by the human SELPGgene, e.g., as described by NCBI RefSeq Gene ID No. 6404. In someembodiments, a tetravalent antibody of the present disclosure binds to ahuman PSGL-1 polypeptide containing 15 or 16 decamer repeats. In someembodiments, a tetravalent antibody of the present disclosure binds to apolypeptide comprising the amino acid sequence of SEQ ID NO:31. In someembodiments, a tetravalent antibody of the present disclosure binds to apolypeptide comprising the amino acid sequence of SEQ ID NO:32. In someembodiments, a tetravalent antibody of the present disclosure binds to apolypeptide comprising the amino acid sequence of SEQ ID NO:31 and bindsto a polypeptide comprising the amino acid sequence of SEQ ID NO:32. Theamino acid sequence of SEQ ID NO:31 depicts full length human PSGL-1,GenBank™ accession number AAA74577.1, GL902797, and the amino acidsequence of SEQ ID NO:32 depicts the shorter 402 amino acid human PSGL-1protein (GenBank™ accession number XP_005269133). In specificembodiments, a tetravalent antibody described herein specifically bindsto human PSGL-1 as determined, e.g., by ELISA or other antigen-bindingassay known in the art, or described herein.

In some embodiments, a VH domain and a VL domain of the presentdisclosure form a VH-VL binding unit (e.g., that specifically binds anepitope, such as an epitope of human PSGL-1). As described herein, aVH-VL binding unit may be formed between a VH domain and a VL domainusing wild-type VH-VL interactions, or a VH-VL binding unit may befurther stabilized using one or more mutations or chemical bonds (e.g.,a disulfide bond, such as the vH44-vL100 disulfide bond introduced bycysteine substitutions in the VH and VL domain of SEQ ID NOs: 29 and 30,respectively).

In some embodiments, a tetravalent antibody of the present disclosurecomprises a dimer of two monomers, where each monomer of the dimercomprises a single-chain polypeptide.

In some embodiments, a single-chain, heavy chain, and/or light chainpolypeptide of the present disclosure comprises a linker sequence. Avariety of linker sequences may suitably be used, e.g., to link VH andVL domains of a VH-VL binding unit, to link a VH or VL domain of a VH-VLbinding unit to a VH or VL domain of another VH-VL binding unit, or tolink a VH or VL domain of a VH-VL binding unit to an antibody constantregion, such as an Fc domain or region. In some embodiments, a linker ofthe present disclosure may be present between domains or regions. Insome embodiments, two domains or regions of the present disclosure maybe joined without a linker, or the linker joining two domains or regionsmay be removed. Coupling of such single-chain fragments using variouslinkers is described in Kortt et al., 1997, Protein Engineering,10:423-433. In some embodiments, a linker sequence of the presentdisclosure comprises 1-50 amino acids. In certain embodiments, a linkersequence of the present disclosure comprises 5-12 amino acids. Exemplarylinker sequences are described herein and illustrated in FIG. 1A. Insome embodiments, a linker sequence of the present disclosure comprisesone or more repeats of the amino acid sequence of GGGGS (SEQ ID NO:25).In some embodiments, a linker sequence of the present disclosurecomprises two, three, four, or five repeats of the amino acid sequenceof GGGGS (SEQ ID NO:25). In some embodiments, a linker sequence of thepresent disclosure comprises the amino acid sequence of SEQ ID NO:33,34, 35, or 36. In some embodiments, a linker sequence of the presentdisclosure comprises the amino acid sequence of GGGGSAAA (SEQ ID NO:26).In some embodiments, a linker sequence of the present disclosurecomprises the amino acid sequence of ASTGS (SEQ ID NO:27). In someembodiments, a linker sequence of the present disclosure comprises theamino acid sequence of ASTGSGGGGS (SEQ ID NO:28).

In some embodiments, a tetravalent antibody of the present disclosurecomprises a dimer of two single-chain diabodies (scDbs), which mayoptionally be fused to an antibody constant region, such as an Fcdomain.

In some embodiments, each monomer of the dimer comprises a single-chainpolypeptide comprising (a) two light chain variable (VL) domains,wherein each of the two VL domains comprises a CDR-L1, a CDR-L2, and aCDR-L3, and wherein the two VL domains are specific for human PSGL-1;(b) two heavy chain variable (VH) domains, wherein each of the two VHdomains comprises a CDR-H1, a CDR-H2, and a CDR-H3, and wherein the twoVH domains are specific for human PSGL-1; and (c) an antibody Fc domain.In some embodiments, each of the two VL domains forms a VH-VL bindingunit with a corresponding VH domain of the two VH domains.

In certain embodiments, each of the two single-chain polypeptidescomprises, from N-terminus to C-terminus: (a) a first VL domain of twoVL domains; (b) a first linker sequence; (c) a first VH domain of two VHdomains; (d) a second linker sequence; (e) a second VL domain of two VLdomains; (f) a third linker sequence; (g) a second VH domain of two VHdomains; (h) a fourth linker sequence; and (i) an antibody Fc domain. Insome embodiments, the first VL domain forms a VH-VL binding unit withthe second VH domain, and the first VH domain forms a VH-VL binding unitwith the second VL domain.

In some embodiments, the first, second and third linker sequences eachcomprise two or more repeats of the amino acid sequence of SEQ ID NO:25.In some embodiments, the first, second or third linker sequencecomprises the amino acid sequence of SEQ ID NO:33, 34, 35, or 36. Insome embodiments, the first and the third linker sequences have the samesequence and comprise two repeats of SEQ ID NO:25. In some embodiments,the second linker sequence comprises five repeats of SEQ ID NO:25. Insome embodiments, the fourth linker sequence comprises the amino acidsequence of SEQ ID NO:26.

In some embodiments, a tetravalent antibody of the present disclosurecomprises a dimer of two tandem single-chain variable fragment (scFv)units (termed taFv for tandem scFv), which may optionally be fused to anantibody constant domain, such as an Fc domain of a heavy chain constantdomain.

In certain embodiments, each of the two single-chain polypeptidescomprises, from N-terminus to C-terminus: (a) a first VH domain of thetwo VH domains; (b) a first linker sequence; (c) a first VL domain ofthe two VL domains; (d) a second linker sequence; (e) a second VL domainof the two VL domains; (f) a third linker sequence; (g) a second VHdomain of the two VH domains; (h) a fourth linker sequence; and (i) anantibody Fc domain. In some embodiments, the first VL domain forms aVH-VL binding unit with the first VH domain, and the second VH domainforms a VH-VL binding unit with the second VL domain. In otherembodiments, each of the two single-chain polypeptides comprises, fromN-terminus to C-terminus: (a) a first VL domain of the two VL domains;(b) a first linker sequence; (c) a first VH domain of the two VHdomains; (d) a second linker sequence; (e) a second VH domain of the twoVH domains; (f) a third linker sequence; (g) a second VL domain of thetwo VL domains; (h) a fourth linker sequence; and (i) the heavy chainconstant domain comprising an antibody Fc domain.

In some embodiments, the first and the third linker sequences have thesame sequence comprising five repeats of SEQ ID NO:25. In someembodiments, the second linker sequence comprises the amino acidsequence of SEQ ID NO:27. In some embodiments, the fourth linkersequence comprises the amino acid sequence of SEQ ID NO:26.

In some embodiments, a tetravalent antibody of the present disclosurecomprises a dimer of two monomers, where each monomer of the dimercomprises an antibody heavy chain and an antibody light chain.

In some embodiments, a tetravalent antibody of the present disclosurecomprises a light chain comprising (i) two light chain variable (VL)domains, wherein each of the two VL domains comprises a CDR-L1, aCDR-L2, and a CDR-L3, and wherein the two VL domains are specific forhuman PSGL-1, (ii) a first heavy chain variable (VH) domain, and (iii) alight chain constant (CL) domain; and/or a heavy chain comprising (i) asecond heavy chain variable (VH) domain, and (ii) a heavy chain constantregion comprising a first heavy chain constant region (CH1) domain, anantibody hinge region, an second heavy chain constant region (CH2)domain, and a third heavy chain constant region (CH3) domain. In someembodiments, the first and the second VH domains each comprise a CDR-H1,a CDR-H2, and a CDR-H3. In some embodiments, the first and the second VHdomains are specific for human PSGL-1. In some embodiments, each of thetwo VL domains forms a VH-VL binding unit with a corresponding VH domainof the first and the second VH domains.

In certain embodiments, the antibody light chain comprises, fromN-terminus to C-terminus: (a) the first VH domain; (b) a first linkersequence; (c) a first VL domain of the two or more VL domains; (d) asecond linker sequence; (e) a second VL domain of the two or more VLdomains; and (f) the CL domain. In some embodiments, the CL domain is akappa CL domain. In other embodiments, the CL domain is a lambda CLdomain. In some embodiments, the first VL domain forms a VH-VL bindingunit with the first VH domain, and the second VH domain forms a VH-VLbinding unit with the second VL domain.

In some embodiments, the first linker sequence comprises five repeats ofSEQ ID NO:25. In some embodiments, the second linker sequence comprisesthe amino acid sequence of SEQ ID NO:28.

In some embodiments, a tetravalent antibody of the present disclosurecomprises a light chain comprising, from N-terminus to C-terminus: (a) afirst VL domain of the two VL domains; (b) the CL domain; (c) a firstlinker sequence; (d) the first VH domain; (e) a second linker sequence;and (f) a second VL domain of the two VL domains. In some embodiments,the CL domain is a kappa CL domain. In other embodiments, the CL domainis a lambda CL domain.

In some embodiments, the first linker sequence comprises two repeats ofSEQ ID NO:25. In some embodiments, the second linker sequence comprisesfive repeats of SEQ ID NO:25.

In some embodiments, a tetravalent antibody of the present disclosurecomprises a heavy chain comprising, from N-terminus to C-terminus: (a)the second of two VH domains; and (b) a heavy chain constant regioncomprising a first heavy chain constant region (CH1) domain, an antibodyhinge region, an second heavy chain constant region (CH2) domain, and athird heavy chain constant region (CH3) domain. In some embodiments, theantibody Fc domain comprises a heavy chain constant 2 (CH2) domain and aheavy chain constant 3 (CH3) domain. In some embodiments, the first VLdomain forms a VH-VL binding unit with the first VH domain, and thesecond VH domain forms a VH-VL binding unit with the second VL domain.

In some embodiments, a tetravalent antibody of the present disclosurecomprises a light chain comprising (i) a first heavy chain variable (VH)domain, (ii) a first light chain variable (VL) domain, and (iii) a lightchain constant (CL) domain; and/or a heavy chain comprising (i) a secondheavy chain variable (VH) domain, (ii) a second light chain variable(VL) domain, and (iii) a heavy chain constant region comprising a firstheavy chain constant region (CH1) domain, an antibody hinge region, ansecond heavy chain constant region (CH2) domain, and a third heavy chainconstant region (CH3) domain. In some embodiments, each of the first andsecond VL domains comprises a CDR-L1, a CDR-L2, and a CDR-L3. In someembodiments, the first and second VL domains are specific for humanPSGL-1. In some embodiments, each of the first and second VH domainscomprises a CDR-H1, a CDR-H2, and a CDR-H3. In some embodiments, thefirst and second VH domains are specific for human PSGL-1. In someembodiments, each of the first and second VL domains forms a VH-VLbinding unit with a corresponding VH domain of the first and second VHdomains.

In some embodiments, the antibody light chain comprises, from N-terminusto C-terminus: (a) the first VH domain; (b) a first linker sequence; (c)the first VL domain; and (d) the CL domain. In some embodiments, the CLdomain is a kappa CL domain. In other embodiments, the CL domain is alambda CL domain.

In some embodiments, the first linker sequence comprises five repeats ofSEQ ID NO:25.

In some embodiments, the antibody heavy chain comprises, from N-terminusto C-terminus: (a) the second of two VH domains; (b) a second linkersequence; (c) the second of two VL domains; and (d) a heavy chainconstant region comprising a first heavy chain constant region (CH1)domain, an antibody hinge region, an second heavy chain constant region(CH2) domain, and a third heavy chain constant region (CH3) domain. Insome embodiments, the antibody Fc domain comprises a heavy chainconstant 2 (CH2) domain and a heavy chain constant 3 (CH3) domain.

In some embodiments, the second linker sequence comprises five repeatsof SEQ ID NO:25.

In some embodiments, a tetravalent antibody of the present disclosurecomprises one or more VH domains comprising one or more CDRs selectedfrom (i) a CDR-H1 comprising the amino acid sequence of SFGMH (SEQ IDNO:17); (ii) a CDR-H2 comprising the amino acid sequence ofYINGGSSTIFYANAVKG (SEQ ID NO:18); and (iii) a CDR-H3 comprising theamino acid sequence of YASYGGGAMDY (SEQ ID NO:19). In some embodiments,a tetravalent antibody of the present disclosure comprises one or moreVH domains comprising (i) a CDR-H1 comprising the amino acid sequence ofSEQ ID NO:17; (ii) a CDR-H2 comprising the amino acid sequence of SEQ IDNO:18; and (iii) a CDR-H3 comprising the amino acid sequence of SEQ IDNO:19. In some embodiments, a tetravalent antibody of the presentdisclosure is a dimer of two monomers, each monomer comprising two VHdomains, each VH domain comprising one or more CDRs selected from (i) aCDR-H1 comprising the amino acid sequence of SFGMH (SEQ ID NO:17); (ii)a CDR-H2 comprising the amino acid sequence of YINGGSSTIFYANAVKG (SEQ IDNO:18); and (iii) a CDR-H3 comprising the amino acid sequence ofYASYGGGAMDY (SEQ ID NO:19). In some embodiments, a tetravalent antibodyof the present disclosure is a dimer of two monomers, each monomercomprising two VH domains, each VH domain comprising (i) a CDR-H1comprising the amino acid sequence of SEQ ID NO:17; (ii) a CDR-H2comprising the amino acid sequence of SEQ ID NO:18; and (iii) a CDR-H3comprising the amino acid sequence of SEQ ID NO:19.

In some embodiments, a tetravalent antibody of the present disclosurecomprises one or more VH domains comprising the amino acid sequence ofSEQ ID NO:23. In some embodiments, a tetravalent antibody of the presentdisclosure comprises a monomer comprising two VH domains, each VH domaincomprising the amino acid sequence of SEQ ID NO:23. In some embodiments,a tetravalent antibody of the present disclosure comprises one or moreVH domains comprising the amino acid sequence of SEQ ID NO:29. In someembodiments, a tetravalent antibody of the present disclosure comprisesa monomer comprising two VH domains, each VH domain comprising the aminoacid sequence of SEQ ID NO:29.

In some embodiments, a tetravalent antibody of the present disclosurecomprises one or more VH domains comprising a sequence having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO:23. In someembodiments, a tetravalent antibody of the present disclosure comprisesa monomer comprising two VH domains, each VH domain comprising asequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% sequence identity to the amino acid sequence of SEQ IDNO:23. In some embodiments, the VH sequence having at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity containssubstitutions (e.g., conservative substitutions), insertions, ordeletions relative to the reference sequence, but an anti-human PSGL-1antibody comprising that sequence retains the ability to bind to humanPSGL-1. In some embodiments, total of 1 to 10 amino acids have beensubstituted, inserted and/or deleted in SEQ ID NO:23.

In some embodiments, a tetravalent antibody of the present disclosurecomprises one or more VH domains comprising a sequence having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO:29. In someembodiments, a tetravalent antibody of the present disclosure comprisesa monomer comprising two VH domains, each VH domain comprising asequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% sequence identity to the amino acid sequence of SEQ IDNO:29. In some embodiments, the VH sequence having at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity containssubstitutions (e.g., conservative substitutions), insertions, ordeletions relative to the reference sequence, but an anti-human PSGL-1antibody comprising that sequence retains the ability to bind to humanPSGL-1. In some embodiments, total of 1 to 10 amino acids have beensubstituted, inserted and/or deleted in SEQ ID NO:29.

In some embodiments, a tetravalent antibody of the present disclosurecomprises one or more VL domains comprising one or more CDRs selectedfrom (i) a CDR-L1 comprising the amino acid sequence of RSSQSIVHNDGNTYFE(SEQ ID NO:20); (ii) a CDR-L2 comprising the amino acid sequence ofKVSNRFS (SEQ ID NO:21); and (iii) a CDR-L3 comprising the amino acidsequence of FQGSYVPLT (SEQ ID NO:22). In some embodiments, a tetravalentantibody of the present disclosure comprises one or more VL domainscomprising (i) a CDR-L1 comprising the amino acid sequence ofRSSQSIVHNDGNTYFE (SEQ ID NO:20); (ii) a CDR-L2 comprising the amino acidsequence of KVSNRFS (SEQ ID NO:21); and (iii) a CDR-L3 comprising theamino acid sequence of FQGSYVPLT (SEQ ID NO:22). In some embodiments, atetravalent antibody of the present disclosure is a dimer of twomonomers, each monomer comprising two VL domains, each VL domaincomprising one or more CDRs selected from (i) a CDR-L1 comprising theamino acid sequence of RSSQSIVHNDGNTYFE (SEQ ID NO:20); (ii) a CDR-L2comprising the amino acid sequence of KVSNRFS (SEQ ID NO:21); and (iii)a CDR-L3 comprising the amino acid sequence of FQGSYVPLT (SEQ ID NO:22).In some embodiments, a tetravalent antibody of the present disclosure isa dimer of two monomers, each monomer comprising two VL domains, each VLdomain comprising (i) a CDR-L1 comprising the amino acid sequence ofRSSQSIVHNDGNTYFE (SEQ ID NO:20); (ii) a CDR-L2 comprising the amino acidsequence of KVSNRFS (SEQ ID NO:21); and (iii) a CDR-L3 comprising theamino acid sequence of FQGSYVPLT (SEQ ID NO:22).

In some embodiments, a tetravalent antibody of the present disclosurecomprises one or more VL domains comprising the amino acid sequence ofSEQ ID NO:24. In some embodiments, a tetravalent antibody of the presentdisclosure comprises a monomer comprising two VL domains, each VL domaincomprising the amino acid sequence of SEQ ID NO:24. In some embodiments,a tetravalent antibody of the present disclosure comprises one or moreVL domains comprising the amino acid sequence of SEQ ID NO:30. In someembodiments, a tetravalent antibody of the present disclosure comprisesa monomer comprising two VL domains, each VL domain comprising the aminoacid sequence of SEQ ID NO:30.

In some embodiments, a tetravalent antibody of the present disclosurecomprises one or more VL domains comprising a sequence having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO:24. In someembodiments, a tetravalent antibody of the present disclosure comprisesa monomer comprising two VL domains, each VL domain comprising asequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% sequence identity to the amino acid sequence of SEQ IDNO:24. In some embodiments, the VL sequence having at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity containssubstitutions (e.g., conservative substitutions), insertions, ordeletions relative to the reference sequence, but an anti-human PSGL-1antibody comprising that sequence retains the ability to bind to humanPSGL-1. In some embodiments, total of 1 to 10 amino acids have beensubstituted, inserted and/or deleted in SEQ ID NO:24.

In some embodiments, a tetravalent antibody of the present disclosurecomprises one or more VL domains comprising a sequence having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO:30. In someembodiments, a tetravalent antibody of the present disclosure comprisesa monomer comprising two VL domains, each VL domain comprising asequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% sequence identity to the amino acid sequence of SEQ IDNO:30. In some embodiments, the VL sequence having at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity containssubstitutions (e.g., conservative substitutions), insertions, ordeletions relative to the reference sequence, but an anti-human PSGL-1antibody comprising that sequence retains the ability to bind to humanPSGL-1. In some embodiments, total of 1 to 10 amino acids have beensubstituted, inserted and/or deleted in SEQ ID NO:30.

In some embodiments, a tetravalent antibody of the present disclosurecomprises a single-chain polypeptide comprising a sequence having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NOs:1, 3, or 5. In someembodiments, a tetravalent antibody of the present disclosure comprisesa dimer of two monomers, each monomer comprising two single-chainpolypeptides, each single-chain polypeptide comprising a sequence havingat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NOs:1, 3, or 5.In some embodiments, the single-chain polypeptide sequence having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identitycontains substitutions (e.g., conservative substitutions), insertions,or deletions relative to the reference sequence, but an anti-humanPSGL-1 antibody comprising that sequence retains the ability to bind tohuman PSGL-1. In some embodiments, total of 1 to 10 amino acids havebeen substituted, inserted and/or deleted in SEQ ID NOs:1, 3, or 5. Insome embodiments, a tetravalent antibody of the present disclosurecomprises two single-chain polypeptides, each comprising the amino acidsequence of SEQ ID NOs:1, 3, or 5.

In some embodiments, a tetravalent antibody of the present disclosurecomprises a single-chain polypeptide comprising a sequence having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to a single-chain polypeptide encoded by the polynucleotidesequence of SEQ ID NOs:2, 4, or 6. In some embodiments, a tetravalentantibody of the present disclosure comprises a dimer of two monomers,each monomer comprising two single-chain polypeptides, each single-chainpolypeptide comprising a sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to asingle-chain polypeptide encoded by the polynucleotide sequence of SEQID NOs:2, 4, or 6. In some embodiments, the single-chain polypeptidesequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity contains substitutions (e.g., conservative substitutions),insertions, or deletions relative to the reference sequence, but ananti-human PSGL-1 antibody comprising that sequence retains the abilityto bind to human PSGL-1. In some embodiments, total of 1 to 10 aminoacids have been substituted, inserted and/or deleted in the single-chainpolypeptide encoded by the polynucleotide sequence of SEQ ID NOs:2, 4,or 6. In some embodiments, a tetravalent antibody of the presentdisclosure comprises two single-chain polypeptides, each encoded by thepolynucleotide sequence of SEQ ID NOs:2, 4, or 6.

In some embodiments, a tetravalent antibody of the present disclosurecomprises a light chain polypeptide comprising a sequence having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NOs:7, 9, or 13. In someembodiments, a tetravalent antibody of the present disclosure comprisesa dimer of two monomers, each monomer comprising a heavy chain and alight chain, and each light chain comprising a sequence having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NOs:7, 9, or 13. In someembodiments, the light chain polypeptide sequence having at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity containssubstitutions (e.g., conservative substitutions), insertions, ordeletions relative to the reference sequence, but an anti-human PSGL-1antibody comprising that sequence retains the ability to bind to humanPSGL-1. In some embodiments, total of 1 to 10 amino acids have beensubstituted, inserted and/or deleted in SEQ ID NOs:7, 9, or 13. In someembodiments, a tetravalent antibody of the present disclosure comprisesa dimer of two monomers, each monomer comprising a heavy chain and alight chain, and each light chain comprising the amino acid sequence ofSEQ ID NOs:7, 9, or 13.

In some embodiments, a tetravalent antibody of the present disclosurecomprises a light chain polypeptide comprising a sequence having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to a light chain polypeptide encoded by the polynucleotidesequence of SEQ ID NOs:8, 10, or 14. In some embodiments, a tetravalentantibody of the present disclosure comprises a dimer of two monomers,each monomer comprising a heavy chain and a light chain, and each lightchain comprising a sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a light chainpolypeptide encoded by the polynucleotide sequence of SEQ ID NOs:8, 10,or 14. In some embodiments, the light chain polypeptide sequence havingat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identitycontains substitutions (e.g., conservative substitutions), insertions,or deletions relative to the reference sequence, but an anti-humanPSGL-1 antibody comprising that sequence retains the ability to bind tohuman PSGL-1. In some embodiments, total of 1 to 10 amino acids havebeen substituted, inserted and/or deleted in the light chain polypeptideencoded by the polynucleotide sequence of SEQ ID NOs:8, 10, or 14. Insome embodiments, a tetravalent antibody of the present disclosurecomprises a dimer of two monomers, each monomer comprising a heavy chainand a light chain, and each light chain comprising a light chainpolypeptide encoded by the polynucleotide sequence of SEQ ID NOs:8, 10,or 14.

In some embodiments, a tetravalent antibody of the present disclosurecomprises a heavy chain polypeptide comprising a sequence having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NOs:11 or 15. In someembodiments, a tetravalent antibody of the present disclosure comprisesa dimer of two monomers, each monomer comprising a heavy chain and alight chain, and each heavy chain comprising a sequence having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NOs:11 or 15. In someembodiments, the heavy chain polypeptide sequence having at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity containssubstitutions (e.g., conservative substitutions), insertions, ordeletions relative to the reference sequence, but an anti-human PSGL-1antibody comprising that sequence retains the ability to bind to humanPSGL-1. In some embodiments, total of 1 to 10 amino acids have beensubstituted, inserted and/or deleted in SEQ ID NOs:11 or 15. In someembodiments, a tetravalent antibody of the present disclosure comprisesa dimer of two monomers, each monomer comprising a heavy chain and alight chain, and each heavy chain comprising the amino acid sequence ofSEQ ID NOs:11 or 15.

In some embodiments, a tetravalent antibody of the present disclosurecomprises a heavy chain polypeptide comprising a sequence having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to a heavy chain polypeptide encoded by the polynucleotidesequence of SEQ ID NOs:12 or 16. In some embodiments, a tetravalentantibody of the present disclosure comprises a dimer of two monomers,each monomer comprising a heavy chain and a light chain, and each heavychain comprising a sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a heavy chainpolypeptide encoded by the polynucleotide sequence of SEQ ID NOs:12 or16. In some embodiments, the heavy chain polypeptide sequence having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identitycontains substitutions (e.g., conservative substitutions), insertions,or deletions relative to the reference sequence, but an anti-humanPSGL-1 antibody comprising that sequence retains the ability to bind tohuman PSGL-1. In some embodiments, total of 1 to 10 amino acids havebeen substituted, inserted and/or deleted in the heavy chain polypeptideencoded by the polynucleotide sequence of SEQ ID NOs:12 or 16. In someembodiments, a tetravalent antibody of the present disclosure comprisesa dimer of two monomers, each monomer comprising a heavy chain and alight chain, and each heavy chain comprising a light chain polypeptideencoded by the polynucleotide sequence of SEQ ID NOs: 12 or 16.

The present disclosure encompasses modifications to antibodies orpolypeptide described herein, including functionally equivalentantibodies which do not significantly affect their properties andvariants which have enhanced or decreased activity and/or affinity.Modification of polypeptides is routine practice in the art and need notbe described in detail herein. Examples of modified polypeptides includepolypeptides with conservative substitutions of amino acid residues, oneor more deletions or additions of amino acids which do not significantlydeleteriously change the functional activity, or use of chemicalanalogs.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue or the antibody fusedto an epitope tag. Other insertional variants of the antibody moleculeinclude the fusion to the N- or C-terminus of the antibody of an enzymeor a polypeptide which increases the serum half-life of the antibody.

Substitution variants have at least one amino acid residue in theantibody molecule removed and a different residue inserted in its place.The sites of greatest interest for substitutional mutagenesis includethe hypervariable regions, but FR alterations are also contemplated.Conservative substitutions are shown in the table below under theheading of “conservative substitutions.” If such substitutions result ina change in biological activity, then more substantial changes,denominated “exemplary substitutions” in the table below, or as furtherdescribed below in reference to amino acid classes, may be introducedand the products screened.

Amino Acid Substitutions

Original Conservative Exemplary Residue Substitutions Substitutions Ala(A) Val Val; Leu; Ile Arg (R) Lys Lys; Gin; Asn Asn (N) Gln Gln; His;Asp, Lys; Arg Asp (D) Glu Glu; Asn Cys (C) Ser Ser; Ala Gln (Q) Asn Asn;Glu Glu (E) Asp Asp; Gln Gly (G) Ala Ala His (H) Arg Asn; Gln; Lys; ArgIle (I) Leu Leu; Val; Met; Ala; Phe; Norleucine Leu (L) Ile Norleucine;Ile; Val; Met; Ala; Phe Lys (K) Arg Arg; Gln; Asn Met (M) Leu Leu; Phe;Ile Phe (F) Tyr Leu; Val; Ile; Ala; Tyr Pro (P) Ala Ala Ser (S) Thr ThrThr (T) Ser Ser Trp (W) Tyr Tyr; Phe Tyr (Y) Phe Trp; Phe; Thr; Ser Val(V) Leu He; Leu; Met; Phe; Ala; Norleucine

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side-chainproperties:

-   -   (1) Non-polar: Norleucine, Met, Ala, Val, Leu, Ile;    -   (2) Polar without charge: Cys, Ser, Thr, Asn, Gln;    -   (3) Acidic (negatively charged): Asp, Glu;    -   (4) Basic (positively charged): Lys, Arg;    -   (5) Residues that influence chain orientation: Gly, Pro; and    -   (6) Aromatic: Trp, Tyr, Phe, His

Non-conservative substitutions are made by exchanging a member of one ofthese classes for another class.

Any cysteine residue not involved in maintaining the proper conformationof the antibody also may be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcross-linking. Conversely, cysteine bond(s) may be added to the antibodyto improve its stability, particularly where the antibody is an antibodyfragment such as an Fv fragment. Exemplary cysteine mutations aredescribed herein (e.g., the G44C VH domain mutation of SEQ ID NO:29, orthe Q100C VL domain mutation of SEQ ID NO:30).

In some embodiments, a tetravalent antibody of the present disclosurecomprises an antibody Fc domain. In some embodiments, the antibody Fcdomain is a human Fc domain. In certain embodiments, the antibody Fcdomain is a human IgG4 Fc domain.

In some embodiments, one or more amino acid residues in the heavy chainconstant region and/or the light chain constant region of the antibodyare modified. For example, amino acid residues of antibodies describedin the Examples may be modified. In some embodiments, the Fc region ofantibodies is modified to enhance or reduce ADCC and/or CDC activitiesof the antibodies. See Shields et al., J. Biol. Chem. 276:6591-6604(2001); Presta et al., Biochem. Soc. Trans. 30:487-490 (2002).

In some embodiments, the Fc region of antibodies is modified to enhancedimer formation and/or stability, or to reduce dimer heterogeneity(e.g., shuffling). It has been demonstrated that a Serine to Prolinemutation at position 241 using Kabat numbering (Kabat et al. 1991,Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242) orat position 228 using the EU index (Edelman et al, 1969, Proc. Natl.Acad. Sci. USA, 63(1): 78-85) in the hinge region of human IgG4 resultsin considerable reduction of intra-chain disulfide bond formation,resulting in the reduction of IgG4 “half-antibody” molecules and reducedheterogeneity/shuffling of IgG4 molecules (Bloom et al. 1997, ProteinSci, 6:407-415; Angal et al, 1993, Molecular Immunology, 30(1):105-108)). There are also published reports that this hinge mutation maydecrease IgG4 shuffling and increase the half-life of the IgG4 moleculesin vivo (Labrijn, et al, 2009, Nat Biotechnol 27:767-771; Stubenrauch,et al, 2010, Drug Metab Dispos 38:84-91). Van der Neut Kolfschoten etal, reported that the C_(H)3 domain of IgG4 and not the core hinge ispredominantly involved in the Fab arm exchange reaction (see Van derNeut Kolfschoten et al, 2007, Science, 317: 1554-1557 (“Van der NeutKolfschoten”) at page 1555, col. 2). Van der Neut Kolfschaten reportedthat exchanging the C_(H)3 domain of IgG1 for the C_(H)3 domain of IgG4activated Fab arm exchange for the IgG1, while exchanging the C_(H)3domain of IgG4 abrogated Fab arm exchange for the IgG4 (see, p. 1555 andFIG. 2D).

In a specific embodiment, provided herein are tetravalent antibodies,that specifically bind to PSGL-1, and that contain one or more aminoacid substitutions in the IgG4 hinge region, wherein said antibody orantigen-binding fragment thereof retains specific binding to said PSGL-1and wherein IgG4 shuffling is reduced relative to an antibody comprisingan IgG4 hinge region not comprising said one or more amino acidsubstitutions. In a specific embodiment, the IgG4 hinge region onlycomprises a single amino acid substitution. An example of a “human IgG4hinge region,” is the region on the heavy chain of an IgG4 antibodybetween the C_(H)1 and C_(H)2 domains, as set forth in Angal et al.,1993, Molecular Immunology, 30(1): 105-108.

In a specific embodiment, a reduction in IgG4 shuffling is determined bydetecting of a lower amount of half antibody molecules or of armexchange produced from an antibody described herein which contains oneor more amino acid substitutions in the hinge region, as compared to theamount of half antibody molecules or of arm exchange produced from anIgG4 molecule containing an IgG4 hinge region not comprising said one ormore amino acid substitutions. Any assay well-known in the art can beused to detect half antibody production and bispecific antibodymolecules. See, e.g., Van der Neut Kolfschoten et al, 2007, Science,317: 1554-1557, for examples of assays to detect production ofbispecific antibodies.

In a specific embodiment, provided herein are tetravalent antibodiesthat specifically bind to PSGL-1 and include a human IgG4 Fc domaincomprising a Serine to Proline amino acid substitution at amino acidposition 228 of the heavy chain numbered according to the EU index (alsoknown as position 241 using Kabat numbering).

Modifications also include glycosylated and nonglycosylatedpolypeptides, as well as polypeptides with other post-translationalmodifications, such as, for example, glycosylation with differentsugars, acetylation, and phosphorylation. Antibodies are glycosylated atconserved positions in their constant regions (Jefferis and Lund, 1997,Chem. Immunol. 65:111-128; Wright and Morrison, 1997, TibTECH 15:26-32).The oligosaccharide side chains of the immunoglobulins affect theprotein's function (Boyd et al., 1996, Mol. Immunol. 32:1311-1318;Wittwe and Howard, 1990, Biochem. 29:4175-4180) and the intramolecularinteraction between portions of the glycoprotein, which can affect theconformation and presented three-dimensional surface of the glycoprotein(Hefferis and Lund, supra; Wyss and Wagner, 1996, Current Opin. Biotech.7:409-416). Oligosaccharides may also serve to target a givenglycoprotein to certain molecules based upon specific recognitionstructures. Glycosylation of antibodies has also been reported to affectantibody-dependent cellular cytotoxicity (ADCC). In particular, CHOcells with tetracycline-regulated expression ofβ(1,4)-N-acetylglucosaminyltransferase III (GnTIII), aglycosyltransferase catalyzing formation of bisecting GlcNAc, wasreported to have improved ADCC activity (Umana et al., 1999, MatureBiotech. 17:176-180).

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine, asparagine-X-threonine, and asparagine-X-cysteine,where X is any amino acid except proline, are the recognition sequencesfor enzymatic attachment of the carbohydrate moiety to the asparagineside chain. Thus, the presence of either of these tripeptide sequencesin a polypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).

The glycosylation pattern of antibodies may also be altered withoutaltering the underlying nucleotide sequence. Glycosylation largelydepends on the host cell used to express the antibody. Since the celltype used for expression of recombinant glycoproteins, e.g., antibodies,as potential therapeutics is rarely the native cell, variations in theglycosylation pattern of the antibodies can be expected (see, e.g., Hseet al., 1997, J. Biol. Chem. 272:9062-9070).

In addition to the choice of host cells, factors that affectglycosylation during recombinant production of antibodies include growthmode, media formulation, culture density, oxygenation, pH, purificationschemes, and the like. Various methods have been proposed to alter theglycosylation pattern achieved in a particular host organism includingintroducing or overexpressing certain enzymes involved inoligosaccharide production (U.S. Pat. Nos. 5,047,335; 5,510,261; and5,278,299). Glycosylation, or certain types of glycosylation, can beenzymatically removed from the glycoprotein, for example usingendoglycosidase H (Endo H), N-glycosidase F, endoglycosidase F1,endoglycosidase F2, or endoglycosidase F3. In addition, the recombinanthost cell can be genetically engineered to be defective in processingcertain types of polysaccharides. These and similar techniques are wellknown in the art.

In some embodiments, an antibody of the present disclosure is modifiedusing coupling techniques known in the art, including, but not limitedto, enzymatic means, oxidative substitution, and chelation.Modifications can be used, for example, for attachment of labels forimmunoassay.

The tetravalent antibody or polypeptide of the present disclosure may beconjugated (for example, linked) to an agent, such as a therapeuticagent or a label. Examples of therapeutic agents are radioactivemoieties, cytotoxins, and chemotherapeutic molecules.

The tetravalent antibody (or polypeptide) of the present disclosure maybe linked to a label such as a fluorescent molecule, a radioactivemolecule, an enzyme, or any other labels known in the art. As usedherein, the term “label” refers to any molecule that can be detected. Ina certain embodiment, an antibody may be labeled by incorporation of aradiolabeled amino acid. In a certain embodiment, biotin moieties thatcan be detected by marked avidin (e.g., streptavidin containing afluorescent marker or enzymatic activity that can be detected by opticalor colorimetric methods) may be attached to the antibody. In certainembodiments, a label may be incorporated into or attached to anotherreagent which in turn binds to the antibody of interest. For example, alabel may be incorporated into or attached to an antibody that in turnspecifically binds the antibody of interest. In certain embodiments, thelabel or marker can also be therapeutic. Various methods of labelingpolypeptides and glycoproteins are known in the art and may be used.Certain general classes of labels include, but are not limited to,enzymatic, fluorescent, chemiluminescent, and radioactive labels.Examples of labels for polypeptides include, but are not limited to, thefollowing: radioisotopes or radionucleoides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S,⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, or ¹³¹I), fluorescent labels (e.g., fluoresceinisothocyanate (FITC), rhodamine, lanthanide phosphors, or phycoerythrin(PE)), enzymatic labels (e.g., horseradish peroxidase, β-galactosidase,luciferase, alkaline phosphatase, glucose oxidase, glucose-6-phosphatedehydrogenase, alcohol dehydrogenase, malate dehydrogenase,penicillinase, or luciferase), chemiluminescent, biotinyl groups,predetermined polypeptide epitopes recognized by a secondary reporter(e.g., leucine zipper pair sequences, binding sites for secondaryantibodies, metal binding domains, or epitope tags). In certainembodiments, labels are attached by spacer arms of various lengths toreduce potential steric hindrance.

Based on the description herein, a tetravalent antibody of the presentdisclosure may be tested according to a variety of in vitro and in vivoassays known in the art. Such assays may include, e.g., binding assaysdirected to the ability of a tetravalent antibody or fragment thereof tobind an epitope or polypeptide of interest (e.g., human PSGL-1 or anepitope thereof), or functional assays directed to one or morefunctional properties of a tetravalent antibody or fragment thereof.

In some embodiments, a tetravalent antibody of the present disclosuremay be tested for binding activity against human PSGL-1. In someembodiments, binding of a tetravalent antibody to human PSGL-1 or anepitope thereof may be tested in an in vitro binding assay. A variety ofbinding assays are known in the art. Such binding assays may becell-based assays (e.g., testing the ability of a tetravalent antibodyto bind a cell expressing human PSGL-1 or an epitope thereof), or theymay be polypeptide-based (e.g., testing the ability of a tetravalentantibody to bind human PSGL-1 or an epitope thereof). In someembodiments, a tetravalent antibody of the present disclosure is testedfor binding to a cell expressing human PSGL-1 (e.g., an Sp2 cell, asexemplified infra) by flow cytometry, FRET, histochemical assays, andthe like. Other suitable binding assays may include without limitationequilibrium methods (e.g., enzyme-linked immunoabsorbent assay (ELISA),radioimmunoassay (RIA), Biacore™ analysis, indirect binding assay,competitive inhibition assay, fluorescence resonance energy transfer(FRET), immunoprecipitation, gel electrophoresis and chromatography(e.g., gel filtration).

In some embodiments, a tetravalent antibody of the present disclosuremay be tested for one or more functional assays for PSGL-1 function. Insome embodiments, a tetravalent antibody of the present disclosure maybe tested for induction of apoptosis in cell(s) expressing human PSGL-1.In some embodiments, a tetravalent antibody of the present disclosuredisplays enhanced induction of apoptosis in a target cell (e.g., a cellexpressing human PSGL-1 or an epitope thereof) as compared to aconventional (e.g., bivalent) antibody having one or more VH or VLdomains in common with the tetravalent antibody (e.g., a parentalantibody). As demonstrated herein, tetravalent antibodies of the presentdisclosure displayed greater potency in inducing apoptosis in targetcells than parental antibodies having a common VH and/or VL domain.Apoptosis assays are described in the art and can be readily carried outby one of skill in the art (see, e.g., Muppidi, J., Porter, M. andSiegel, R. M. 2004. Measurement of Apoptosis and Other Forms of CellDeath. Current Protocols in Immunology. 59:3.17.1-3.17.36). Selectedassays for detecting apoptosis (e.g., Annexin V or propidium iodidestaining) are exemplified supra. The term “induce” or “inducing” meansinitiation of or an increase of apoptosis above a control level.Apoptosis of activated T cells can be induced by about 10%, about 20%,about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about90%, about 100%, about 125%, about 150% or more compared to a control(e.g. Apoptosis of activated T cells in the absence of the antibodiesdescribe herein or in the presence of a non-specific antibody).

T cells and T cell lines which are appropriate for use in the assaysdescribed herein relating to PSGL-1 activity are readily available(e.g., ARR, DU.528, Jurkat, H-SB2, RPMI 8402, CML-T1, Karpas 45,KE-37/SKW-3, SUP-T1, SUP-T3, MOLT 3/4, P12-Ichikawa, PF-382, CCRF-CEM,HPB-ALL, K-Tl, TALL-1, MOLT 16/17, TALL-104, DND-41, Loucy, MOLT 13,Peer/Bel3, HUT 78/H9, HUT 102, MT-1, DEL, JB6, Karpas 299, SU-DHL1,12H5, 3D054.8, 3DO11.10, 8D051.15, or 3D018.3) or can be readilyidentified using methods known in the art (see, e.g., Thornton, A. M.2003. Fractionation of T and B Cells Using Magnetic Beads. CurrentProtocols in Immunology. 55:3.5A. 1-3.5A. i l., Hathcock, K. 2001. TCell Enrichment by Cytotoxic Elimination of B Cells and Accessory Cells.Current Protocols in Immunology. 00:3.3.1-3.3.5., Horgan, K., Shaw, S.and Boirivant, M. 2009. Immunomagnetic Purification of T CellSubpopulations. Current Protocols in Immunology. 85:7.4.1-7.4.9., andKanof, M. E. 2001. Purification of T Cell Subpopulations. CurrentProtocols in Immunology. 00:7.3.1-7.3.5). In particular embodiments,cells or cell lines for use in cell proliferation assays can expressPSGL-1, endogenously or recombinantly. Cells or cell lines for use incell viability assays can express PSGL-1, endogenously or recombinantly,and exert changes in cell viability in response to PSGL-1 ligand oranti-PSGL-1 antibody binding. Cells or cell lines for use in apoptosisassays can express PSGL-1, endogenously or recombinantly, and exertchanges in apoptosis in response to PSGL-1 ligand or anti-PSGL-1antibody binding. Preferably the cells or cell lines are human (e.g.ARR, DU.528, Jurkat, H-SB2, RPMI 8402, CML-Tl, Karpas 45, KE-37/SKW-3,SUP-Tl, SUP-T3, MOLT 3/4, P12-Ichikawa, PF-382, CCRF-CEM, HPB-ALL, K-Tl,TALL-1, MOLT 16/17, TALL-104, DND-41, Loucy, MOLT 13, Peer/Bel3, HUT78/H9, HUT 102, MT-1, DEL, JB6, Karpas 299, or SU-DHL1).

In some embodiments, a tetravalent antibody of the present disclosuremay be tested for inhibition of delayed type hypersensitivity (DTH). Insome embodiments, a tetravalent antibody of the present disclosuredisplays enhanced inhibition of DTH (e.g., in a trans vivo animal model)as compared to a conventional (e.g., bivalent) antibody having one ormore VH or VL domains in common with the tetravalent antibody (e.g., aparental antibody). As demonstrated herein, tetravalent antibodies ofthe present disclosure displayed greater potency in inhibiting DTH in atrans vivo mouse footpad swelling model than parental antibodies havinga common VH and/or VL domain. DTH assays are described in the art andexemplified infra and can be readily carried out by one of skill in theart. In some embodiments, a tetravalent antibody of the presentdisclosure may display a potency of DTH inhibition that may be increasedby about 10%, about 20%, about 30%, about 40%, about 50%, about 60%,about 70%, about 80%, about 90%, about 100%, about 125%, about 150%,about 200%, about 300%, about 400%, about 500%, about 600%, or morecompared to a control (e.g. inhibition of DTH by a conventional orbivalent antibody, such as the parental antibody).

III. Polynucleotides, Vectors, Host Cells, and Antibody Production

The present disclosure also provides polynucleotides comprising apolynucleotide encoding any of the tetravalent antibodies and/orpolypeptides described herein. In some embodiments, the polypeptidescomprise the sequences of light chain and heavy chain variable regions.In some embodiments, the polynucleotide is an isolated polynucleotide(e.g., isolated from a host cell or from one or more differentpolynucleotides).

Provided herein are polynucleotides encoding any of the tetravalentantibodies or polypeptide constituents (e.g., monomers such assingle-chain polypeptides, antibody heavy chains, and/or antibody lightchains) described herein. In some embodiments, a polynucleotide of thepresent disclosure encodes a polypeptide sequence selected from SEQ IDNOs:1, 3, 5, 7, 9, 11, 13, 15, and 17-31. In some embodiments, apolynucleotide of the present disclosure comprises a polynucleotidesequence selected from SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, and 16. Insome embodiments, a polynucleotide of the present disclosure comprisesone or more introns. In other embodiments, a polynucleotide of thepresent disclosure does not comprise an intron, e.g., a cDNA orprocessed mRNA sequence.

It is appreciated by those of ordinary skill in the art that, as aresult of the degeneracy of the genetic code, there are many nucleotidesequences that encode a polypeptide as described herein. Some of thesepolynucleotides bear minimal homology to the nucleotide sequence of anynative gene. Thus, polynucleotides that vary due to differences in codonusage are specifically contemplated by the present disclosure. Further,alleles of the genes comprising the polynucleotide sequences providedherein are within the scope of the present disclosure. Alleles areendogenous genes that are altered as a result of one or more mutations,such as deletions, additions, and/or substitutions of nucleotides. Theresulting mRNA and protein can, but need not, have an altered structureor function. Alleles can be identified using standard techniques (suchas hybridization, amplification, and/or database sequence comparison).

Also provided herein are polynucleotides that are optimized, e.g., bycodon/RNA optimization, replacement with heterologous signal sequences,and elimination of mRNA instability elements. Methods to generateoptimized nucleic acids encoding a tetravalent antibody or polypeptidethereof for recombinant expression by introducing codon changes and/oreliminating inhibitory regions in the mRNA can be carried out byadapting the optimization methods described in, e.g., U.S. Pat. Nos.5,965,726; 6, 174,666; 6,291,664; 6,414, 132; and 6,794,498,accordingly. For example, potential splice sites and instabilityelements (e.g., A/T or A/U rich elements) within the RNA can be mutatedwithout altering the amino acids encoded by the nucleic acid sequencesto increase stability of the RNA for recombinant expression. Thealterations utilize the degeneracy of the genetic code, e.g., using analternative codon for an identical amino acid. In some embodiments, itcan be desirable to alter one or more codons to encode a conservativemutation, e.g., a similar amino acid with similar chemical structure andproperties and/or function as the original amino acid. Such methods canincrease expression of an anti-PSGL-1 tetravalent antibody orpolypeptide thereof relative to the expression of an anti-PSGL-1tetravalent antibody or polypeptide thereof encoded by polynucleotidesthat have not been optimized. Furthermore, the polynucleotide sequencescan be designed to match the preferred codon usage in the host cell,e.g. E. coli codon usage or CHO codon usage.

An optimized polynucleotide sequence encoding a tetravalent antibody orpolypeptide thereof described herein can hybridize to an unoptimizedpolynucleotide sequence encoding a tetravalent antibody or polypeptidethereof described herein. In specific embodiments, an optimizednucleotide sequence encoding a tetravalent antibody or polypeptidethereof described herein hybridizes under high stringency conditions toan unoptimized polynucleotide sequence encoding a tetravalent antibodyor polypeptide thereof described herein. In a specific embodiment, anoptimized nucleotide sequence encoding a tetravalent antibody orpolypeptide thereof described herein hybridizes under high stringency,intermediate or lower stringency hybridization conditions to anunoptimized nucleotide sequence encoding a tetravalent antibody orpolypeptide thereof described herein. Information regardinghybridization conditions have been described, see, e.g., U.S. PatentApplication Publication No. US 2005/0048549 (e.g., paragraphs 72-73),which is incorporated herein by reference in its entirety.

The polynucleotides of the present disclosure can be obtained usingchemical synthesis, recombinant methods, or PCR. Methods of chemicalpolynucleotide synthesis are well known in the art and need not bedescribed in detail herein. One of skill in the art can use thesequences provided herein and a commercial DNA synthesizer to produce adesired DNA sequence.

For preparing polynucleotides using recombinant methods, apolynucleotide comprising a desired sequence can be inserted into asuitable vector, and the vector in turn can be introduced into asuitable host cell for replication and amplification, as furtherdiscussed herein. Polynucleotides can be inserted into host cells by anymeans known in the art. Cells are transformed by introducing anexogenous polynucleotide by direct uptake, endocytosis, transfection,F-mating, or electroporation. Once introduced, the exogenouspolynucleotide can be maintained within the cell as a non-integratedvector (such as a plasmid) or integrated into the host cell genome. Thepolynucleotide so amplified can be isolated from the host cell bymethods well known within the art. See, e.g., Sambrook et al. (1989).

Alternatively, PCR allows reproduction of DNA sequences. PCR technologyis well known in the art and is described in U.S. Pat. Nos. 4,683,195;4,800,159; 4,754,065; and 4,683,202, as well as PCR: The PolymeraseChain Reaction, Mullis et al. eds., Birkauswer Press, Boston (1994).

The present disclosure also provides vectors (e.g., cloning vectors orexpression vectors) comprising a nucleic acid sequence encoding any ofthe polypeptides (including antibodies) described herein. Suitablecloning vectors can be constructed according to standard techniques ormay be selected from a large number of cloning vectors available in theart. While the cloning vector selected may vary according to the hostcell intended to be used, useful cloning vectors generally have theability to self-replicate, may possess a single target for a particularrestriction endonuclease, and/or may carry genes for a marker that canbe used in selecting clones containing the vector. Suitable examplesinclude plasmids and bacterial viruses, e.g., pUC18, pUC19, Bluescript(e.g., pBS SK+) and its derivatives, mp18, mp19, pBR322, pMB9, ColE1,pCR1, RP4, phage DNAs, and shuttle vectors such as pSA3 and pAT28. Theseand many other cloning vectors are available from commercial vendorssuch as BioRad, Strategene, and Invitrogen.

Expression vectors generally are replicable polynucleotide constructsthat contain a polynucleotide according to the present disclosure. Theexpression vector may replicable in the host cells either as episomes oras an integral part of the chromosomal DNA. Suitable expression vectorsinclude but are not limited to plasmids, viral vectors, includingadenoviruses, adeno-associated viruses, retroviruses, cosmids, andexpression vector(s) disclosed in PCT Publication No. WO 87/04462.Vector components may generally include, but are not limited to, one ormore of the following: a signal sequence; an origin of replication; oneor more marker genes; and suitable transcriptional controlling elements(such as promoters, enhancers, or terminator). For expression (i.e.,translation), one or more translational controlling elements are alsousually required, such as ribosome binding sites, translation initiationsites, or stop codons.

Methods of making antibodies and polypeptides derived from theantibodies are known in the art and are disclosed herein.Well-established methods may be used to identify anti-PSGL antibodies(e.g., antibodies that specifically bind to human PSGL-1), from whichvariable domains (e.g., VH and/or VL domains) may be used in thetetravalent antibodies of the present disclosure. Exemplary anti-humanPSGL-1 antibodies, as well as methods for screening, producing, andpurifying such antibodies, are described in International ApplicationPub. No. WO 2012/174001.

Additional anti-human PSGL-1 antibodies may be identified using methodsknown in the art, such as those described in International ApplicationPub. No. WO 2012/174001 and supra. For example, the monoclonalantibodies can be prepared using hybridoma technology, such as thosedescribed by Kohler and Milstein (1975), Nature, 256:495. In a hybridomamethod, a mouse, a hamster, or other appropriate host animal, istypically immunized with an immunizing agent (e.g., a cell expressinghuman PSGL-1 or a fragment thereof) to elicit lymphocytes that produceor are capable of producing antibodies that specifically bind to theimmunizing agent. Alternatively, the lymphocytes may be immunized invitro. The lymphocytes are then fused with an immortalized cell lineusing a suitable fusing agent, such as polyethylene glycol, to form ahybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice,Academic Press, (1986) pp. 59-1031). Immortalized cell lines are usuallytransformed mammalian cells, particularly myeloma cells of rodent,rabbit, bovine, or human origin. Usually, rat or mouse myeloma celllines are employed. The hybridoma cells may be cultured in a suitableculture medium that desirably contains one or more substances thatinhibit the growth or survival of the unfused, immortalized cells. Forexample, if the parental cells lack the enzyme hypoxanthine guaninephosphoribosyl transferase (HGPRT or HPRT), the culture medium for thehybridomas typically includes hypoxanthine, aminopterin, and thymidine(“HAT medium”), which substances prevent the growth of HGPRT-deficientcells.

Desired immortalized cell lines are those that fuse efficiently, supportstable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More desirable immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies (Kozbor, J. Immunol. (1984), 133:3001; Brodeur etal., Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63).

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies. The antibody may bescreened for having specific binding to an ORP150 polypeptide (such asbinding to an epitope in an extracellular domain of the ORP150polypeptide) obtained from or expressed on the cell surface ofplasmacytoma, multiple myeloma, colorectal, gastric, or esophagealcancer or tumor cells. Cancer cells or an ORP150 polypeptide (or afragment thereof containing an extracellular domain of an ORP150polypeptide) may be used for screening. For example, RPMI8226, U266,NCI-H929, L363, Colo205, DLD-1, HT29, SNU-1, Kato-III, or CE146T cellsmay be used for screening. A polypeptide comprising amino acids 673-800,701-800, 673-752, or 723-732 of SEQ ID NO:17 may also be used forscreening.

In some embodiments, the binding specificity of monoclonal antibodiesproduced by the hybridoma cells is determined by immunoprecipitation orby an in vitro binding assay, such as radioimmunoassay (RIA) orenzyme-linked immunoabsorbent assay (ELISA). Such techniques and assaysare known in the art. The binding affinity of the monoclonal antibodycan, for example, be determined by the Scatchard analysis of Munson andPollard (1980), Anal. Biochem., 107:220.

After the desired hybridoma cells are identified, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, supra). Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium or RPMI-1640 medium.Alternatively, the hybridoma cells may be grown in vivo as ascites in amammal.

The monoclonal antibodies can be generated by culturing the hybridomacells, and the antibodies secreted by the hybridoma cells may further beisolated or purified. Antibodies may be isolated or purified from theculture medium or ascites fluid by conventional immunoglobulinpurification procedures such as, for example, protein A-Sepharose,hydroxylapatite chromatography, gel electrophoresis, dialysis, oraffinity chromatography.

The tetravalent antibodies or polypeptides of the present disclosure maybe generated by screening a library of antibodies or polypeptides toselect antibodies or polypeptides that bind to human PSGL-1, e.g.,expressed on the cell surface of a cell. Antibody phage displaylibraries known in the art may be used. In some embodiments, theantibodies in the library (e.g., displayed on phage) are single-chain Fv(scFv) fragments or Fab fragment. In some embodiments, the antibodies inthe library (e.g., displayed on phage) are single-domain antibodies. Forexample, a single-domain antibody may comprise all or a portion of theheavy chain variable domain or all or a portion of the light chainvariable domain of an antibody. In some embodiments, the antibodies inthe library are human antibodies. The antibodies identified may furtherbe tested for their capabilities to induce cell death (e.g., apoptosis)and/or bind human PSGL-1 using methods known in the art and describedherein.

The tetravalent antibodies of the present disclosure can be made byrecombinant DNA methods, such as those described in U.S. Pat. Nos.4,816,567 and 6,331,415. For example, DNA encoding the variable orconstant region of any of the tetravalent antibodies of the presentdisclosure (or single, heavy, or light chain polypeptides that areconstituents thereof) can be readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of murine antibodies). The hybridoma cells of the presentdisclosure serve as a preferred source of such DNA. Once isolated, theDNA can be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein to synthesize monoclonal antibodies in the recombinant hostcells. The DNA also can be modified, for example, by substituting thecoding sequence for human heavy and light chain constant domains inplace of the homologous murine sequences (U.S. Pat. No. 4,816,567) or bycovalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulin polypeptide. Such anon-immunoglobulin polypeptide can be substituted for the constantdomains of an antibody of the present disclosure, or can be substitutedfor the variable domains of one antigen-combining site of an antibody ofthe present disclosure to create a chimeric bivalent antibody.

In some embodiments, the tetravalent antibodies of the presentdisclosure are expressed from two expression vectors. For example, eachexpression vector may express one monomer of a dimer of the presentdisclosure (e.g., a single-chain polypeptide or antibody heavy or lightchain polypeptide). Alternatively, both monomers of a dimer of thepresent disclosure are expressed from a single expression vector.

Normally the expression vector has transcriptional and translationalregulatory sequences which are derived from a species compatible with ahost cell. In addition, the vector ordinarily carries a specific gene(s)which is (are) capable of providing phenotypic selection in transformedcells.

A wide variety of recombinant host-vector expression systems foreukaryotic cells are known and can be used in the present disclosure.For example, Saccharomyces cerevisiae, or common baker's yeast, is themost commonly used among eukaryotic microorganisms, although a number ofother strains, such as Pichia pastoris, are available. Cell linesderived from multicellular organisms such as Sp2/0 or Chinese HamsterOvary (CHO), which are available from the ATCC, may also be used ashosts. Typical vector plasmids suitable for eukaryotic celltransformations are, for example, pSV2neo and pSV2gpt (ATCC), pSVL andpSVK3 (Pharmacia), and pBPV-1/pML2d (International Biotechnology, Inc.).

The eukaryotic host cells useful in the present disclosure are, forexample, hybridoma, myeloma, plasmacytoma, or lymphoma cells. However,other eukaryotic host cells may be suitably utilized provided themammalian host cells are capable of recognizing transcriptional andtranslational DNA sequences for expression of the proteins; processingthe leader peptide by cleavage of the leader sequence and secretion ofthe proteins; and providing post-translational modifications of theproteins, e.g., glycosylation.

Accordingly, the present disclosure provides host cells (e.g.,eukaryotic host cells) which are transformed by recombinant expressionvectors comprising DNA constructs disclosed herein and which are capableof expressing the tetravalent antibodies or polypeptides of the presentdisclosure. In some embodiments, the transformed host cells of thepresent disclosure comprise at least one DNA construct comprising apolynucleotide of the present disclosure, or a polynucleotide expressinga monomer, dimer, or tetravalent antibody of the present disclosure, andtranscriptional and translational regulatory sequences which arepositioned in relation to the coding DNA sequences to direct expressionof antibodies or polypeptides.

Any host cells capable of over-expressing heterologous DNAs can be usedfor the purpose of isolating the genes encoding the antibody,polypeptide, or protein of interest. Non-limiting examples of mammalianhost cells include but not limited to COS, HeLa, and CHO cells. See alsoPCT Publication No. WO 87/04462. Suitable non-mammalian host cellsinclude prokaryotes (such as E. coli or B. subtilis) and yeast (such asS. cerevisae, S. pombe, or K. lactis).

The host cells used in the present disclosure may be transformed in avariety of ways by standard transfection procedures well known in theart. Among the standard transfection procedures which may be used areelectroporation techniques, protoplast fusion and calcium-phosphateprecipitation techniques. Such techniques are generally described by F.Toneguzzo et al. (1986), Mol. Cell. Biol., 6:703-706; G. Chu et al.,Nucleic Acid Res. (1987), 15:1311-1325; D. Rice et al., Proc. Natl.Acad. Sci. USA (1979), 79:7862-7865; and V. Oi et al., Proc. Natl. Acad.Sci. USA (1983), 80:825-829. The vectors containing the polynucleotidesof interest can be introduced into the host cell by any of a number ofappropriate means, including electroporation, transfection employingcalcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, orother substances; microprojectile bombardment; lipofection; andinfection (e.g., where the vector is an infectious agent such asvaccinia virus). The choice of introducing vectors or polynucleotidesoften depends on features of the host cell.

In the case of two expression vectors, the two expression vectors can betransferred into a host cell one by one separately or together(co-transfer or co-transfect).

The present disclosure also provides a method for producing theantibodies or polypeptides that comprises culturing a host cellcomprising an expression vector(s) encoding the antibodies or thepolypeptides, and recovering the antibodies or polypeptides from theculture by ways well known to one skilled in the art.

Furthermore, the desired antibodies can be produced in a transgenicanimal. A suitable transgenic animal can be obtained according tostandard methods which include micro-injecting into eggs the appropriateexpression vectors, transferring the eggs into pseudo-pregnant females,and selecting a descendant expressing the desired antibody.

The present disclosure also provides chimeric tetravalent antibodiesthat specifically bind human PSGL-1. For example, the variable andconstant regions of the tetravalent antibody are from separate species.In some embodiments, the variable regions of both heavy chain and lightchain are from the murine antibodies described herein. The chimericantibody of the present disclosure can be prepared by techniqueswell-established in the art. See for example, U.S. Pat. Nos. 6,808,901;6,652,852; 6,329,508; 6,120,767; and 5,677,427, each of which is herebyincorporated by reference. In general, the chimeric antibody can beprepared by obtaining cDNAs encoding the heavy and light chain variableregions of the antibodies, inserting the cDNAs into an expressionvector, which upon being introduced into eukaryotic host cells,expresses the chimeric antibody of the present disclosure. Desirably,the expression vector carries a functionally complete constant heavy orlight chain sequence so that any variable heavy or light chain sequencecan be easily inserted into the expression vector.

The present disclosure provides a humanized tetravalent antibody thatspecifically binds to human PSGL-1. The humanized antibody is typicallya human antibody in which residues from CDRs are replaced with residuesfrom CDRs of a non-human species such as mouse, rat, or rabbit havingthe desired specificity, affinity and capacity. In some instances, Fvframework residues of the human antibody are replaced by correspondingnon-human residues.

There are four general steps to humanize a monoclonal antibody. Theseare: (1) determining the nucleotide and predicted amino acid sequence ofthe starting antibody light and heavy variable domains, (2) designingthe humanized antibody, i.e., deciding which antibody framework regionto use during the humanizing process, (3) the actual humanizingmethodologies/techniques, and (4) the transfection and expression of thehumanized antibody. See, for example, U.S. Pat. Nos. 4,816,567;5,807,715; 5,866,692; 6,331,415; 5,530,101; 5,693,761; 5,693,762;5,585,089; 6,180,370; and 6,548,640. For example, the constant regionmay be engineered to more resemble human constant regions to avoidimmune response if the antibody is used in clinical trials andtreatments in humans. See, for example, U.S. Pat. Nos. 5,997,867 and5,866,692.

It is important that antibodies be humanized with retention of highaffinity for the antigen and other favorable biological properties. Toachieve this goal, humanized antibodies can be prepared by a process ofanalysis of the parental sequences and various conceptual humanizedproducts using three dimensional models of the parental and humanizedsequences. Three dimensional immunoglobulin models are commonlyavailable and are familiar to those skilled in the art. Computerprograms are available which illustrate and display probablethree-dimensional conformational structures of selected candidateimmunoglobulin sequences. Inspection of these displays permits analysisof the likely role of the residues in the functioning of the candidateimmunoglobulin sequence, i.e., the analysis of residues that influencethe ability of the candidate immunoglobulin to bind its antigen. In thisway, FR residues can be selected and combined from the consensus andimport sequence so that the desired antibody characteristic, such asincreased affinity for the target antigen(s), is achieved. In general,the CDR residues are directly and most substantially involved ininfluencing antigen binding. The humanized antibodies may also containmodifications in the hinge region to improve one or more characteristicsof the antibody.

In another alternative, antibodies may be screened and maderecombinantly by phage display technology. See, for example, U.S. Pat.Nos. 5,565,332; 5,580,717; 5,733,743 and 6,265,150; and Winter et al.,Annu. Rev. Immunol. 12:433-455 (1994). Alternatively, the phage displaytechnology (McCafferty et al., Nature 348:552-553 (1990)) can be used toproduce human antibodies and antibody fragments in vitro, fromimmunoglobulin variable (V) domain gene repertoires from unimmunizeddonors. According to this technique, antibody V domain genes are clonedin-frame into either a major or minor coat protein gene of a filamentousbacteriophage, such as M13 or fd, and displayed as functional antibodyfragments on the surface of the phage particle. Because the filamentousparticle contains a single-stranded DNA copy of the phage genome,selections based on the functional properties of the antibody alsoresult in selection of the gene encoding the antibody exhibiting thoseproperties. Thus, the phage mimics some of the properties of the B-cell.Phage display can be performed in a variety of formats; for review see,e.g., Johnson, Kevin S. and Chiswell, David J., Current Opinion inStructural Biology 3, 564-571 (1993). Several sources of V-gene segmentscan be used for phage display. Clackson et al., Nature 352:624-628(1991) isolated a diverse array of anti-oxazolone antibodies from asmall random combinatorial library of V genes derived from the spleensof immunized mice. A repertoire of V genes from unimmunized human donorscan be constructed, and antibodies to a diverse array of antigens(including self-antigens) can be isolated essentially following thetechniques described by Mark et al., J. Mol. Biol. 222:581-597 (1991),or Griffith et al., EMBO J. 12:725-734 (1993). In a natural immuneresponse, antibody genes accumulate mutations at a high rate (somatichypermutation). Some of the changes introduced will confer higheraffinity, and B-cells displaying high-affinity surface immunoglobulinare preferentially replicated and differentiated during subsequentantigen challenge. This natural process can be mimicked by employing thetechnique known as “chain shuffling.” Marks et al., Bio/Technol.10:779-783 (1992)). In this method, the affinity of “primary” humanantibodies obtained by phage display can be improved by sequentiallyreplacing the heavy and light chain V region genes with repertoires ofnaturally occurring variants (repertoires) of V domain genes obtainedfrom unimmunized donors. This technique allows the production ofantibodies and antibody fragments with affinities in the pM-nM range. Astrategy for making very large phage antibody repertoires (also known as“the mother-of-all libraries”) has been described by Waterhouse et al.,Nucl. Acids Res. 21:2265-2266 (1993). Gene shuffling can also be used toderive human antibodies from rodent antibodies, where the human antibodyhas similar affinities and specificities to the starting rodentantibody. According to this method, which is also referred to as“epitope imprinting,” the heavy or light chain V domain gene of rodentantibodies obtained by phage display technique is replaced with arepertoire of human V domain genes, creating rodent-human chimeras.Selection on antigen results in isolation of human variable regionscapable of restoring a functional antigen-binding site, i.e., theepitope governs (imprints) the choice of partner. When the process isrepeated in order to replace the remaining rodent V domain, a humanantibody is obtained (see PCT Publication No. WO 93/06213, publishedApr. 1, 1993). Unlike traditional humanization of rodent antibodies byCDR grafting, this technique provides completely human antibodies, whichhave no framework or CDR residues of rodent origin. It is apparent thatalthough the above discussion pertains to humanized antibodies, thegeneral principles discussed are applicable to customizing antibodiesfor use, for example, in dogs, cats, primates, equines, and bovines.

In certain embodiments, the antibody is a fully human antibody.Non-human antibodies that specifically bind an antigen can be used toproduce a fully human antibody that binds to that antigen. For example,the skilled artisan can employ a chain swapping technique, in which theheavy chain of a non-human antibody is co-expressed with an expressionlibrary expressing different human light chains. The resulting hybridantibodies, containing one human light chain and one non-human heavychain, are then screened for antigen binding. The light chains thatparticipate in antigen binding are then co-expressed with a library ofhuman antibody heavy chains. The resulting human antibodies are screenedonce more for antigen binding. Techniques such as this one are furtherdescribed in U.S. Pat. No. 5,565,332. In addition, an antigen can beused to inoculate an animal that is transgenic for human immunoglobulingenes. See, e.g., U.S. Pat. No. 5,661,016.

The present disclosure also provides bispecific antibodies. A bispecificantibody has binding specificities for at least two different antigens(including different epitopes). In some embodiments, a bispecificantibody of the present disclosure includes two or more different VHand/or VL domains that specifically bind PSGL-1. In some embodiments,the two or more different VH and/or VL domains specifically bind thesame epitope of PSGL-1. In some embodiments, the two or more differentVH and/or VL domains specifically bind different epitopes of PSGL-1,which may or may not be overlapping epitopes.

A bispecific antibody (a monoclonal antibody that has bindingspecificities for at least two different antigens) can be prepared usingthe antibodies disclosed herein. Methods for making bispecificantibodies are known in the art (see, e.g., Suresh et al., 1986, Methodsin Enzymology 121:210). Traditionally, the recombinant production ofbispecific antibodies was based on the coexpression of twoimmunoglobulin heavy chain-light chain pairs, with the two heavy chainshaving different specificities (Millstein and Cuello, 1983, Nature 305,537-539). In some embodiments, a bispecific tetravalent antibody may beproduced using the methods exemplified supra.

According to one approach to making bispecific antibodies, antibodyvariable domains with the desired binding specificities(antibody-antigen combining sites) are fused to immunoglobulin constantdomain sequences. In some embodiments, the fusion is with animmunoglobulin heavy chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. In some embodiments, the first heavychain constant region (CH1), containing the site necessary for lightchain binding, is present in at least one of the fusions. DNAs encodingthe immunoglobulin heavy chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are cotransfected into a suitable host organism. Thisprovides for great flexibility in adjusting the mutual proportions ofthe three polypeptide fragments in embodiments when unequal ratios ofthe three polypeptide chains used in the construction provide theoptimum yields. It is, however, possible to insert the coding sequencesfor two or all three polypeptide chains in one expression vector whenthe expression of at least two polypeptide chains in equal ratiosresults in high yields or when the ratios are of no particularsignificance.

Heteroconjugate antibodies, comprising two covalently joined monomers orantibodies, are also within the scope of the present disclosure. Suchantibodies have been used to target immune system cells to unwantedcells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection (PCTPublication Nos. WO 91/00360 and WO 92/200373; and EP 03089).Heteroconjugate antibodies may be made using any convenientcross-linking methods. Suitable cross-linking agents and techniques arewell known in the art, and are described in U.S. Pat. No. 4,676,980.

Certain aspects of the present disclosure relate to antibody variabledomains and/or antibody fragments, e.g., that may be used as aconstituent of a tetravalent antibody described herein. Antibodyfragments may contain the active binding region of the antibodies, suchas Fab, F(ab′)₂, scFv, Fv fragments, and the like. Various methods knownin the art may be used to produce and/or isolate antibody fragments,which may be incorporated into a tetravalent antibody of the presentdisclosure, e.g., by standard recombinant techniques known in the artbased on the concepts described herein.

Single-chain Fv fragments may be produced, such as described in Iliadeset al., 1997, FEBS Letters, 409:437-441. Coupling of such single-chainfragments using various linkers is described in Kortt et al., 1997,Protein Engineering, 10:423-433. A variety of techniques for therecombinant production and manipulation of antibodies are well known inthe art. Such fragments can be produced from the monoclonal antibodiesdescribed herein using techniques well established in the art (Rousseauxet al. (1986), in Methods Enzymol., 121:663-69 Academic Press).

Methods of preparing antibody fragments are well known in the art. Forexample, an antibody fragment can be produced by enzymatic cleavage ofantibodies with pepsin to provide a 100 Kd fragment denoted F(ab′)₂.This fragment can be further cleaved using a thiol reducing agent, andoptionally a blocking group for the sulfhydryl groups resulting fromcleavage of disulfide linkages, to produce 50 Kd Fab′ monovalentfragments. Alternatively, an enzymatic cleavage using papain producestwo monovalent Fab fragments and an Fc fragment directly. These methodsare described, for example, by U.S. Pat. Nos. 4,036,945 and 4,331,647and references contained therein, which patents are incorporated hereinby reference. Also, see Nisonoff et al. (1960), Arch Biochem. Biophys.89: 230; Porter (1959), Biochem. J. 73: 119; Smyth (1967), Methods inEnzymology 11: 421-426. Alternatively, the Fab can be produced byinserting DNA encoding Fab of the antibody into an expression vector forprokaryote or an expression vector for eukaryote, and introducing thevector into a prokaryote or eukaryote to express the Fab.

IV. Methods and Uses

Certain aspects of the present disclosure relate to methods and uses forthe tetravalent antibodies described herein. These methods and uses arebased at least in part on the properties of the tetravalent antibodiesas described herein, including without limitation their increased numberof epitope binding domains, potential for lesser dependence uponcross-linking in vitro and/or in vivo, differential potency for inducingapoptosis (e.g., of human PSGL-1 expressing cells), and/or enhanced invivo or trans vivo efficacy.

As described herein, PSGL-1 is known to be involved in inflammation andT cell biology. The tetravalent antibodies of the present disclosurethat specifically bind human PSGL-1 may find use, inter alia, intreating individuals with diseases related to T cell function (e.g., aT-cell mediated inflammatory disease), or individuals in need of medicalprocedures that may result in inflammatory conditions such asimmunological reactions, or for which such conditions are managedbeforehand (e.g., a transplantation or transfusion).

In some embodiments, a disorder or disease treated by the methodsdescribed herein may be a T-cell mediated inflammatory disease.Non-limiting examples of disorders and diseases that can be treated, orone or more of whose symptoms may be ameliorated or prevented using thetetravalent antibodies described herein described herein includepsoriasis, Crohn's disease, ankylosing spondylitis, arthritis (includingrheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, andpsoriatic arthritis), diabetes mellitus, multiple sclerosis,encephalomyelitis, myasthenia gravis, systemic lupus erythematosus,autoimmune thyroiditis, dermatitis (including atopic dermatitis andeczematous dermatitis), Sjogren's Syndrome, aphthous ulcer, iritis,conjunctivitis, keratoconjunctivitis, type I diabetes, inflammatorybowel diseases, ulcerative colitis, asthma, allergic asthma, cutaneouslupus erythematosus, scleroderma, vaginitis, proctitis, drug eruptions,leprosy reversal reactions, erythema nodosum leprosum, autoimmuneuveitis, allergic encephalomyelitis, acute necrotizing hemorrhagicencephalopathy, idiopathic bilateral progressive sensorineural hearingloss, aplastic anemia, pure red cell anemia, idiopathicthrombocytopenia, polychondritis, Wegener's granulomatosis, chronicactive hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichenplanus, Graves' disease, graft versus host disease (GVHD), sarcoidosis,primary biliary cirrhosis, uveitis posterior, interstitial lungfibrosis, allergies such as atopic allergy, AIDS, and T cell neoplasmssuch as leukemias or lymphomas. In some embodiments, the disease is anautoimmune disease.

In another embodiment, the disease or disorder treated in accordancewith the methods described herein is plaque psoriasis. Plaque psoriasisor psoriasis vulgaris is the most common form of psoriasis and ischaracterized by sharply demarcated, raised erythematous skin plaquescovered by silvery scale. There is a predilection of the lesions toinvolve the extensor surfaces of the extremities, the lumbosacral area,and the scalp. The corresponding histopathological findings includesignificant inflammatory cellular infiltration of the dermis andepidermis, increased numbers of dilated vessels, and a substantialthickening of the epidermis with disordered differentiation ofkeratinocytes and hyperkeratosis. Approximately one third of patientswith plaque psoriasis are categorized as having moderate or severedisease and are consequently candidates for therapy beyond just topicaltreatment.

In another embodiment, the disorder treated in accordance with themethods described herein is chronic plaque psoriasis. Symptoms of plaquechronic psoriasis include, but are not limited to, single or multipleraised reddened patches of skin, ranging from coin-sized to larger, onany part of the body, including but not limited to the knees, elbows,lumbosacral regions, scalp, and nails.

In another embodiment, the disorder treated in accordance with themethods described herein is guttate psoriasis. Symptoms of guttatepsoriasis include, but are not limited to, flares of water drop shapedscaly plaques on the skin, followed by an infection, such as astreptococcal throat infection.

In another embodiment, the disease or disorder treated in accordancewith the methods described herein is inverse psoriasis. Symptoms ofinverse psoriasis include, but are not limited to, smooth, usually moistareas of skin that are red and inflamed, unlike the scaling associatedwith plaque psoriasis, on one or more of the following body parts:armpits, groin, under the breasts, and in other skin folds around thegenitals and buttocks.

In another embodiment, the disease or disorder treated in accordancewith the methods described herein is pustular psoriasis. Symptoms ofpustular psoriasis include, but are not limited to, pus-filled blistersthat vary in size and location, but mostly on the hands and feet.

In another embodiment, the disease or disorder treated in accordancewith the methods described herein is erythodermic psoriasis. Symptoms oferythodermic psoriasis include, but are not limited to, periodic,widespread, fiery redness of the skin and the shedding of scales insheets, rather than smaller flakes. The reddening and shedding of theskin are often accompanied by severe itching and pain, heart rateincrease, and fluctuating body temperature.

In another embodiment, the disease or disorder treated in accordancewith the methods described herein is rheumatoid arthritis. Symptoms ofrheumatoid arthritis, include, but are not limited to, fatigue, loss ofappetite, low fever, swollen glands, weakness, joint pain in wrists,elbows, shoulders, hips, knees, ankles, toes, jaw, hands, feet, fingers,and/or neck, morning stiffness, chest pain when taking a breath(pleurisy), eye burning, itching, and discharge, nodules under the skin,numbness, tingling, or burning in the hands and feet.

In another embodiment, the disease or disorder treated in accordancewith the methods described herein is Crohn's disease. Symptoms ofCrohn's disease, but are not limited to, crampy abdominal (belly area)pain, fever, fatigue, loss of appetite, pain with passing stool(tenesmus), persistent, watery diarrhea, unintentional weight loss,constipation, eye inflammation, fistulas (usually around the rectalarea, may cause draining of pus, mucus, or stools), joint pain, liverinflammation, mouth ulcers, rectal bleeding and bloody stools, skinlumps or sores (ulcers), and swollen gums.

In another embodiment, the disease or disorder treated in accordancewith the methods described herein is ankylosing spondylitis. Symptoms ofankylosing spondylitis include, but are not limited to, frequent painand stiffness in the lower back and buttocks, spine, and/or neck; andpain and tenderness spreading to the ribs, shoulder blades, hips, thighsand heels; inflammation of the eye (iridocyclitis and uveitis), causingredness, eye pain, vision loss, floaters and photophobia; fatigue; andnausea.

In another embodiment, the disease or disorder treated in accordancewith the methods described herein is diabetes mellitus. Symptoms ofdiabetes mellitus include, but are not limited to, loss of weight,polyuria (frequent urination), polydipsia (increased thirst), polyphagia(increased hunger), cardiovascular disease, diabetic retinopathy,diabetic neuropathy, hyperosmolar nonketotic state, and diabeticketoacidosis.

In some embodiments, a tetravalent antibody or composition of thepresent disclosure may be administered to the individual before,concurrently with, and/or after a transplantation. For example, asdescribed in greater detail below, a tetravalent antibody or compositionof the present disclosure may be administered to increase the likelihoodof a favorable treatment outcome, decrease the likelihood of anunfavorable outcome, and/or mitigate or prevent symptoms or unfavorableoutcomes occurring before, concurrently with, or after thetransplantation has been completed.

As used herein, treating an individual in need of a transplantation mayrefer to one or more of therapeutic treatment and prophylactic orpreventative measures (e.g., increasing the likelihood of a favorabletreatment outcome, such as graft survival, graft function, or decreasingthe likelihood of an unfavorable outcome, such as an unfavorableresponse to treatment, or a condition that reduces the likelihood afavorable treatment, such as a transplantation, from occurring).Treating may include without limitation mitigating or preventingconditions and symptoms associated with a disorder or a condition,and/or problems or conditions that interfere with or limit anindividual's access to treatment options of a disorder or a condition,such as sensitization, hypersensitization, high panel reactiveantibodies (PRA) level and/or presence of pre-existing alloantibodiesthat limit availability of grafts to an individual awaiting atransplantation. Those in need of treatment include those already withthe disorder or condition, as well as those in which the disorder orcondition is to be prevented. Treatment of a disorder or condition maysuppress immune-mediated events associated with the disorder orcondition, ameliorate the symptoms of the disorder or condition, reducethe severity of the disorder or condition, alter the course of thedisorder or condition progression, and/or ameliorate or cure the basicdisorder or condition.

For example, successful treatment of an individual awaitingtransplantation include, but is not limited to, reducing the level ofalloantibodies, reducing panel reactive antibodies (PRA), enabling theindividual to have more cross-match compatible donors, increasing thelikelihood or probability of the individual to receive a graft,shortening the expected waiting period of the individual for a graft,desensitizing the individual, lowering risk of transplant-associatedsymptoms or conditions (such as immune-mediated events as describedbelow), or any combination thereof.

For example, successful treatment of an individual receiving atransplantation includes, but is not limited to, protection andmaintenance of the transplanted organ or tissue for a long term, whichcomprises controlling, reversing, mitigating, delaying, or preventingone or more symptoms or undesirable conditions associated with the organtransplant, such as immune-mediated events, including, but not limitedto, production of donor-specific alloantibodies (DSA), GVHD,antibody-mediated rejection (AMR), hyperacute graft rejection, chronicgraft rejection, graft failure, and graft loss, as measured byfunctional or histological signs of the symptom or condition. Atreatment capable of controlling a disorder or condition (e.g., graftrejection) may include a treatment that slows the progression of thedisease process, when initiated after functional or histological signsof the disorder or condition (e.g., graft rejection) are observed.Further, a treatment capable of reversing a disease or condition (e.g.,graft rejection) may include a treatment that, when initiated afterfunctional or histological signs of the disease or condition (e.g.,graft rejection) have appeared, reverses the disease process and returnsfunctional and histological findings closer to normal. A treatmentcapable of “delaying progression” of a disorder or condition (e.g.,graft rejection) may include deferring, hindering, slowing, retarding,stabilizing, and/or postponing development of the disorder or condition(e.g., graft rejection). This delay can be of varying lengths of time,depending on the history of the disease and/or individual being treated.As is evident to one skilled in the art, a sufficient or significantdelay can, in effect, encompass prevention, in that the individual,e.g., an individual at risk for developing the disorder or condition,does not develop the disorder or condition.

In some embodiments, a transplantation of the present disclosure may betransplantation of one or more tissues or organs including withoutlimitation bone marrow, kidney, heart, liver, neuronal tissue, lung,pancreas, skin, and intestine (e.g., small and/or large intestine, aswell as any sub-tissues thereof).

In addition, tetravalent antibodies are useful for preventing and/ortreating certain disorders and diseases associated with or caused (inwhole or in part) by increased proliferation and/or numbers of activatedT cells relative to the proliferation and/or numbers of activated Tcells found in healthy individuals or individuals not having theparticular disorder or disease. Non-limiting examples of disorders anddiseases that can be prevented and/or treated using the tetravalentantibodies described herein include graft-versus-host disease and casesof transplantation rejection (including transplantation rejection usingallogeneic or xenogeneic tissues) such as bone marrow transplantation,liver transplantation, kidney transplant, or the transplantation of anyorgan or tissue.

In some embodiments, a tetravalent antibody or composition of thepresent disclosure may be administered to the individual before,concurrently with, and/or after a transfusion. For example, as describedin greater detail below, a tetravalent antibody or composition of thepresent disclosure may be administered to increase the likelihood of afavorable treatment outcome, decrease the likelihood of an unfavorableoutcome, and/or mitigate or prevent symptoms occurring before,concurrently with, or after the transfusion has been completed.

As used herein, treating an individual in need of a transfusion mayrefer to one or more of therapeutic treatment and prophylactic orpreventative measures (e.g., increasing the likelihood of a favorabletreatment outcome, such as replacement or supplementation of bloodcomponents/cells, or decreasing the likelihood of an unfavorableoutcome, such as an unfavorable response to treatment, inefficacy oftreatment, or immunological reaction, or a condition that reduces thelikelihood a favorable treatment, such as a transfusion, fromoccurring). Treating may include without limitation mitigating orpreventing conditions and symptoms associated with a disorder or acondition, and/or problems or conditions that interfere with or limit anindividual's access to treatment options of a disorder or a condition.Those in need of treatment include those already with the disorder orcondition, as well as those in which the disorder or condition is to beprevented. Treatment of a disorder or condition may suppressimmune-mediated events associated with the disorder or condition,ameliorate the symptoms of the disorder or condition, reduce theseverity of the disorder or condition, alter the course of the disorderor condition progression, and/or ameliorate or cure the basic disorderor condition.

In some embodiments, the transfusion is a transfusion comprising one ormore of white blood cells, red blood cells, and platelets. In someembodiments, the transfusion comprises whole blood or one or more bloodproducts, including without limitation white blood cells, red bloodcells, platelets, fresh frozen plasma, cryoprecipitate or blood clottingfactors, antibodies, and/or blood substitutes. Exemplary conditions thatmay be treated with a transfusion (e.g., transfusion of blood or a bloodproduct) include without limitation hemorrhage or blood loss, reducedhematocrit or hemoglobin (e.g., anemia), sickle cell disease,thalassemia, blood supplementation during or after surgical procedures,cardiac disease, traumatic injury, deficiency of one or more bloodfactors (e.g., hemophilia, von Willebrand disease, hypofibrinogenemia,or a deficiency in factor II, V, VII, IX, X, or XI), conditionsrequiring fibrinogen supplementation (e.g., liver disease, bloodtransfusion, etc.), bone marrow failure, platelet function disorders,thrombocytopenia, immunodeficiency (e.g., from a therapy or disease),and the like. Descriptions of practices, dosing, responses, indications,and preparations related to transfusions may be found, e.g., in theAmerican Red Cross Compendium of Transfusion Practice Guidelines.

Administration of a tetravalent antibody or polypeptide in accordancewith the methods described herein can be continuous or intermittent,depending, for example, upon the recipient's physiological condition,whether the purpose of the administration is therapeutic orprophylactic, and other factors known to skilled practitioners. Theadministration of an antibody or a polypeptide may be essentiallycontinuous over a preselected period of time or may be in a series ofspaced dose.

The dosage and frequency of administration of a tetravalent antibodydescribed herein or a pharmaceutical composition thereof is administeredin accordance with the methods for preventing and/or treating whileminimizing side effects. The exact dosage of a tetravalent antibodydescribed herein to be administered to a particular subject or apharmaceutical composition thereof can be determined by a practitioner,in light of factors related to the subject that requires treatment.Factors which can be taken into account include the severity of thedisease state, general health of the subject, age, and weight of thesubject, diet, time and frequency of administration, combination(s) withother therapeutic agents or drugs, reaction sensitivities, andtolerance/response to therapy. The dosage and frequency ofadministration of a tetravalent antibody described herein or apharmaceutical composition thereof can be adjusted over time to providesufficient levels of the antibody or an antibody derived antigen-bindingfragment, or to maintain the desired effect.

The precise dose to be employed in the formulation will also depend onthe route of administration, and the seriousness of an inflammatorydisorder or disease, and should be decided according to the judgment ofthe practitioner and each patient's circumstances.

Effective doses can be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems.

In one embodiment, any of the compositions described herein isformulated for administration by intraperitoneal, intravenous,subcutaneous, or intramuscular injections, or other forms ofadministration such as oral, mucosal, via inhalation, sublingually, etc.Parenteral administration, in one embodiment, is characterized byinjection, either subcutaneously, intramuscularly or intravenously isalso contemplated herein. Injectables can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution or suspension in liquid prior to injection, or asemulsions. The injectables, solutions and emulsions also contain one ormore excipients. Suitable excipients are, for example, water, saline,dextrose, glycerol or ethanol. In addition, if desired, thepharmaceutical compositions to be administered can also contain minoramounts of non-toxic auxiliary substances such as wetting or emulsifyingagents, pH buffering agents, stabilizers, solubility enhancers, andother such agents. Other routes of administration may include, entericadministration, intracerebral administration, nasal administration,intraarterial administration, intracardiac administration, intraosseousinfusion, intrathecal administration, intravenous infusion, subcutaneousimplantation or injection, intramuscular administration, intrarectaladministration intravaginal administration, intragastricaladministration, intratracheal administration, intrapulmonaryadministration and intraperitoneal administration. Preparations forparenteral administration include sterile solutions ready for injection,sterile dry soluble products, such as lyophilized powders, ready to becombined with a solvent just prior to use, including sterile suspensionsready for injection, sterile dry insoluble products ready to be combinedwith a vehicle just prior to use and sterile emulsions. The solutionscan be either aqueous or nonaqueous. If administered intravenously,suitable carriers include physiological saline or phosphate bufferedsaline (PBS), water, and solutions containing thickening andsolubilizing agents, such as glucose, polyethylene glycol, andpolypropylene glycol and mixtures thereof.

In another embodiment, the present disclosure also contemplatesadministration of a composition comprising the antibodies orpolypeptides of the present disclosure conjugated to other molecules,such as detectable labels, or therapeutic or cytotoxic agents. Theagents may include, but are not limited to radioisotopes, toxins,toxoids, inflammatory agents, enzymes, antisense molecules, peptides,cytokines, and chemotherapeutic agents. Methods of conjugating theantibodies with such molecules are generally known to those of skilledin the art. See, e.g., PCT publications WO 92/08495; WO 91/14438; WO89/12624; U.S. Pat. No. 5,314,995; and EP 396,387.

In one embodiment, the composition comprises an antibody or polypeptideconjugated to a cytotoxic agent. Cytotoxic agents can include any agentsthat are detrimental to cells. An exemplary class of cytotoxic agentsthat can be conjugated to the antibodies or fragments may include, butare not limited to, paclitaxol, cytochalasin B, gramicidin D, ethidiumbromide, emetine, mitomycin, etoposide, teniposide, vincristine,vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol,puromycin, and analogs or homologs thereof.

V. Pharmaceutical Compositions

The present disclosure also provides pharmaceutical compositionscomprising tetravalent antibodies or polypeptides described herein, anda pharmaceutically acceptable carrier or excipients. The pharmaceuticalcompositions may find use, e.g., in the methods, uses, and/or kits ofthe present disclosure.

Pharmaceutically acceptable carriers or excipients are known in the art,and are relatively inert substances that facilitate administration of apharmacologically effective substance. For example, an excipient cangive form or consistency, or act as a diluent. Suitable excipientsinclude but are not limited to stabilizing agents, wetting andemulsifying agents, salts for varying osmolarity, encapsulating agents,buffers, and skin penetration enhancers. In certain embodiments, atetravalent antibody described herein is in a liquid pharmaceuticalcomposition. Liquid pharmaceutically administrable compositions can, forexample, be prepared by dissolving, dispersing, or otherwise mixing anantibody described herein in a carrier, such as, for example, water,saline, aqueous dextrose, glycerol, glycols, ethanol, and the like, tothereby form a solution or suspension. If desired, the pharmaceuticalcomposition to be administered can also contain minor amounts ofnontoxic auxiliary substances such as wetting agents, emulsifyingagents, solubilizing agents, and pH buffering agents and the like.Excipients as well as formulations for parenteral and nonparenteral drugdelivery are set forth in Remington, The Science and Practice ofPharmacy 20th Ed. Mack Publishing (2000).

The pharmaceutical compositions are provided for administration tohumans and animals in unit dosage forms, such as sterile parenteralsolutions or suspensions containing suitable quantities of a tetravalentantibody described herein. The tetravalent antibody is, in oneembodiment, formulated and administered in unit-dosage forms ormultiple-dosage forms. Unit-dose forms as used herein refers tophysically discrete units suitable for human and animal subjects andpackaged individually as is known in the art. Each unit-dose contains apredetermined quantity of the antibody or the antibody derivedantigen-binding fragment sufficient to produce the desired therapeuticeffect, in association with the required pharmaceutical carrier, vehicleor diluent. Examples of unit-dose forms include ampoules and syringes.Unit-dose forms can be administered in fractions or multiples thereof. Amultiple-dose form is a plurality of identical unit-dosage formspackaged in a single container to be administered in segregatedunit-dose form. Examples of multiple-dose forms include vials, orbottles of pints or gallons. Hence, multiple dose form is a multiple ofunit-doses which are not segregated in packaging.

The concentration of tetravalent antibody in the pharmaceuticalcomposition will depend on, e.g., the physicochemical characteristics ofthe antibody or the antibody derived antigen-binding fragment, thedosage schedule, and amount administered as well as other factors knownto those of skill in the art. In some embodiments, the pharmaceuticalcompositions provide a dosage of from about 0.001 mg to about 100 mg oftetravalent antibody per kilogram of body weight per day. Pharmaceuticaldosage unit forms can be prepared to provide from about 0.001 mg toabout 100 mg, and/or a combination of other optional essentialingredients per dosage unit form.

In some embodiments, the present disclosure provides tetravalentantibodies and compositions (such as the pharmaceutical compositionsdescribed herein) for use in any of the methods described herein,whether in the context of use as a medicament and/or use for manufactureof a medicament.

VI. Kits

Certain aspects of the present disclosure are related to kits orarticles of manufacture that comprise a tetravalent antibody of thepresent disclosure. Optionally, the kits described herein may containone or more pharmaceutically acceptable carriers, such as the exemplarycarriers described herein. In some embodiments, a kit of the presentdisclosure includes a pharmaceutical composition of the presentdisclosure. Kits described herein may find use, e.g., in the methods oruses of the present disclosure.

Kits may optionally provide additional components such as buffers andinterpretive information. Normally, the kit comprises a container and alabel or package insert(s) on or associated with the container. Thecontainers may be unit doses, bulk packages (e.g., multi-dose packages)or sub-unit doses. Instructions supplied in the kits of the presentdisclosure are typically written instructions on a label or packageinsert (e.g., a paper sheet included in the kit), but machine-readableinstructions (e.g., instructions carried on a magnetic or opticalstorage disk) are also acceptable.

In some embodiments, the kits further include a package insertcomprising instructions for administration of the tetravalent antibodyto treat a T-cell mediated inflammatory disease. In some embodiments,the kits further include a package insert comprising instructions foradministration of the tetravalent antibody before, concurrently with,and/or after a transfusion or transplantation.

The kits of the present disclosure are in suitable packaging. Suitablepackaging includes, but is not limited to, vials, bottles, jars,flexible packaging (e.g., sealed Mylar or plastic bags), and the like.Also contemplated are packages for use in combination with a specificdevice, such as an inhaler, nasal administration device (e.g., anatomizer), or an infusion device such as a minipump. A kit may have asterile access port (for example the container may be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). The container may also have a sterile access port(for example the container may be an intravenous solution bag or a vialhaving a stopper pierceable by a hypodermic injection needle). At leastone active agent in the composition is a tetravalent antibody orpolypeptide described herein. The container may further comprise asecond pharmaceutically active agent. In some embodiments, a kit mayfurther include any other material or device useful in a treatment(e.g., a transfusion or transplantation), including without limitationone or more containers, tubing, sterilizing agents or equipment,cannulae, syringes, and the like.

EXAMPLES

The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the invention. It is understood that the examples andembodiments described herein are for illustrative purposes only and thatvarious modifications or changes in light thereof will be suggested topersons skilled in the art and are to be included within the spirit andpurview of this application and scope of the appended claims.

Example 1: Generation and Characterization of Anti-PS GL-1 TetravalentAntibodies

P-selectin Glycoprotein Ligand-1 (PSGL-1) is expressed on a wide rangeof hematopoietic cells, including myeloid, lymphoid, dendritic, andCD34+ stem cell populations (see, e.g., Spertini et al. 1996, J CellBiol. 135(2):523-31). Several mouse antibodies specific for PSGL-1 andcapable of inducing apoptosis in T cells have previously beenidentified. Among these mouse antibodies, an antibody (h15A7) that didnot interfere with the interaction between P-selectin and PSGL-1, whichrequired for efficient localization of T cells and neutrophils to targetinflammatory tissues, was chosen for clinical development and wasmodified to a humanized kappa-light-chain containing IgG4 monoclonalantibody to minimize ADCC and CDC on PSGL-1 expressing cells (see, e.g.,U.S. Pat. No. 7,604,800). Subsequently, h15A7 was further engineered toproduce h15A7H, which has a mutation of SER228PRO in hinge region ofh15A7 (International Application Pub. No. WO 2012/174001). This mutationwas introduced in order to reduce antibody shuffling, the intermolecularexchange among IgG4 antibodies in vivo. In vitro studies showed thath15A7/h15A7H preferentially induced apoptosis of late-stage activated Tcells but not other PSGL-1-expressing cells. Without wishing to be boundto theory, it is thought that the mechanism of action of h15A7H appearsto be dependent at least in part on cross-linking of human PSGL-1molecules, which is mediated by antibody cross-linker in vitro andpossibly FcR-expressing cells in vivo.

The Example presented below describes the development of severalcross-linker/FcR-expressing cell-independent tetravalent antibodiesderived from h15A7H (FIGS. 1A & 1B). Without wishing to be bound totheory, tetravalent antibodies may possess advantages over h15A7H forclinical development, e.g., treatment of T-cell mediated inflammatorydiseases. These results demonstrate that tetravalent h15A7H antibodiesshow enhanced efficacy compared to the parental h15A7H antibody both invitro and trans vivo.

Methods

Cells and Reagents

Sp2/0-Ag14 (ATCCC®L-1581™) and Sp2/0-hPSGL-1 were cultured in 90% DMEM(GIBCO®, Cat. No. 11965-092™) supplemented with 10% FBS (GIBCO′, Cat.No. 26140-079), 100 U/mL penicillin/100 μg/mL streptomycin (GIBCO®, Cat.No. 15140) and 1 mM sodium pyruvate (GIBCO®, Cat. No. 11360).

The h15A7H antibody was described in International Application Pub. No.WO 2012/174001. The h15A7H tetravalent antibodies used in the study wereproduced from a Flp-In CHO stable cell line, purified by protein Aaffinity chromatography, and maintained in Dulbecco's Phosphate-BufferedSaline (GIBCO® Cat. No. 21600-069)/0.02% Tween-20 (JT Baker® X251-07).Human IgG4p/K as irrelevant isotype control antibody was produced fromFlp-In CHO cells. 12H5.5 is a murine IgG1 anti-idiotype antibody againsth15A7/h15A7H.

Animals

Female B6 mice at 6-8 weeks of age were obtained from BioLASCO TaiwanCo., Ltd, Taipei, Taiwan. All mice were maintained under specificpathogen-free conditions. All animal studies were conducted followingthe guidelines of the Institutional Animal Care and Use Committee.

Construction of Anti-PSGL-1 Tetravalent Antibody Variants

scDb₂-Fc

scDb₂-Fc (FIG. 1A, left) included 2 domains of single-chain diabodies(scDbs) fused in parallel to the N-terminals of human IgG4 Fc with amutation in the hinge region to minimize half-antibody exchange in vivo.Each scDb domain contained not only a domain sequence of VL-VH-VL-VH,but also a linker (G₄S₁)₅ (SEQ ID NO:33) between VH and VL and twoidentical linkers (e.g., SEQ ID NO:34) between VL and VH. SeveralscDb-Fcs with said linkers of different length were generated foroptimization

taFv₂-Fc

taFv₂-Fc (FIG. 1A, middle) included 2 tandem single-chain variablefragment (scFv) units (termed taFv for tandem scFv) fused in parallel tothe N-terminals of human IgG4 Fc with a mutation in the hinge region tominimize half-antibody exchange in vivo. There were three differentkinds of scFvs used to construct taFv, including v2 (VH-VL), v3 (VL-VH),and v4 (VL-VH) versions, containing a linker (G₄S₁)₅ (SEQ ID NO:33)between VH and VL. Among them, v2 and v4 were structure-constrained bythe formation of VH44-VL100 disulfide bond. The VH44-VL100 disulfidebond was introduced into scFv in both VL-VH and VH-VL orientations forincreased conformational stability (see SEQ ID NOs:29 and 30). Each taFvhad either sequential v2-v3 or sequential v4-v2 of anti-PSGL-1 scFv witha linker ASTGS (SEQ ID NO:27) between the two scFvs.

scFv-IgG

The disulfide-constrained v2 version of anti-PSGL-1 scFv was used togenerate 3 scFv-IgG4p variants (FIG. 1A, right), includingscFv₄-crIgG4p, scFv₂-LC-IgG4p, and LC-scFv₂-IgG4p. scFv₄-crIgG4p had 4scFv units fused in parallel to the N-terminals of both constant regionsof kappa light chain and heavy chain of IgG4p (crIgG) without a linker.scFv₂-LC-IgG4p had 2 scFv units fused in parallel to the N-terminals ofkappa light chains of h15A7H IgG with a linker ASTGSG₄S (SEQ ID NO:28)in-between, whereas LC-scFv₂-IgG4p had 2 scFv units fused in parallel tothe C-terminals of kappa light chains of h15A7H IgG with a linker (G₄S)₂(SEQ ID NO:34) in-between. Light chains of LC-scFv₂ IgG4p and scFv₂-LCIgG4p formats were separately sub-cloned into a pcDNA5/FRT vector thatencoded an intact h15A7H heavy chain sequence for antibody expression.FIG. 1B shows another diagram of these tetravalent antibody formats withthe variable fragments shaded.

cDNAs of all tetravalent antibodies were cloned into the pcDNA5/FRTvector (Invitrogen™, Cat. No: V6010-20) for tetravalent antibodyexpression.

Production of Stable Cell Lines Expressing Anti-PSGL-1 TetravalentAntibody Variants

Anti-PSGL1 tetravalent antibody variants were stably expressed andproduced in Flp-In CHO cells (Invitrogen™, Cat. No: R708-07). The cDNAsequences of tetravalent antibody variants were inserted into thepcDNA5/FRT vector (Invitrogen™, Cat. No: V6010-20) and cotransfectedwith pOG44 (Invitrogen®, Cat. No V6005-20) following the standardprocedure provided by the vendor. The culture supernatants of theestablished cell lines were collected and purified with protein Asepharose heads (GE Healthcare™, Cat. No: 17-5280-04). The purifiedproteins were analyzed with both SDS-PAGE and size exclusionchromatography to ensure the quality of antibodies.

Reducing and Non-Reducing SDS-PAGE (Sodium Dodecyl SulfatePolyacrylamide Gel Electrophoresis)

Purified anti-PSGL-1 tetravalent antibodies were electrophoresed in 10%reducing and non-reducing SDS polyacrylamide gels. For the reducing SDSpolyacrylamide gels, 2 μg of antibody were mixed with 5×SDS samplebuffer (300 nM Tris, pH6.8, 10% SDS, 50% glycerol, 5% 2-mercaptoethanoland 0.06% bromophenol blue) and boiled for 10 min at 100° C. beforeloading. For the non-reducing SDS polyacrylamide gels, 2 μg ofantibodies were mixed with 5× non-reducing sample buffer (300 nM Tris,pH6.8, 10% SDS, 50% glycerol and 0.06% bromophenol blue) and boiled for10 min at 100° C. before loading. The reducing and non-reducing proteinsamples were loaded onto the same SDS-polyacrylamide gels whereelectrophoresis was performed. Coomassie blue staining was used todetect proteins on the gel after electrophoresis.

Binding Assay of Anti-PSGL-1 Tetravalent Antibody Variants

Sp2/0 cells transfected with human PSGL-1(Sp2/0-hPSGL1) were used as thePSGL-1 expressing cell line. Sp2/0-hPSGL1 cells were centrifuged at 1200rpm for 5 min. The cell pellets were resuspended in FACS buffer (PBScontaining 1% FBS) and pipetted into 96 well plate (1×10⁵ cells/well).To each well was added 1000 of supernatants containing humanized15A7H(h15A7H)/tetravalent antibodies, and these were incubated for 60min at 4° C. The cells were washed three times with cold FACS buffer andthen incubated with 1000 of Mouse Anti-Human IgG4 pFc′-PE(SouthernBiotech Cat. no. 9190-09) at 1 μg/ml concentration for 60 minat 4° C. Subsequently, the cells were washed three times with cold FACSbuffer and analyzed by FACS analysis. All flow cytometric analyses wereperformed on a BD-LSR flow cytometer (Becton Dickinson) using the CellQuest software.

Apoptosis Assay of Anti-PSGL-1 Tetravalent Antibody Variants

1×10⁵ Sp2/0-hPSGL1 cells were seeded into the wells of 96-well plates.Aliquots of purified anti-PSGL-1 tetravalent and control antibodies attitrated concentrations were prepared freshly and added to each well.The treated cells were kept at 37° C. for 6 hr before FACS analysis forcellular apoptosis assay.

For the cellular apoptosis assay, an Annexin-V-FITC Apoptosis DetectionKit (Strong Biotech, Cat. No. AVK250) was used following themanufacturer's instructions. In brief, the treated cells were harvestedand resuspended in 100 μl Annexin V binding buffer containing 0.5 μlAnnexin V-FITC at room temperature. After 15 min incubation in the dark,the cells were washed twice with 200 μl of Annexin V binding buffer.Before FACS analysis, 1 μl of propidium iodide (PI) per sample wasadded. All flow cytometric analyses were performed on a BD-LSR flowcytometer (Becton Dickinson) using Cell Quest software. The Annexin Vpositive and/or PI positive cells are considered apoptotic cells.

Isolation of Human Peripheral Blood Mononuclear Cells (PBMCs)

500 ml whole blood was collected from healthy donors that werepreviously tested as good tetanus responders. The blood was centrifugedat 1500 rpm for 6 min. The upper plasma layer was discarded, and theremnant blood was diluted with an equivalent volume of PBS. The dilutedwhole blood was carefully added over a Ficoll (GE, Ficoll Plaque Plus,Cat #17-1440-02) layer and centrifuged at 2400 rpm for 15 mins at roomtemperature. The buffy coat layer containing mononuclear cells wascollected and washed with PBS 3 times to minimize plateletcontamination. The cells were resuspended in PBS and kept on ice beforeuse.

Trans-Vivo Delayed Type Hypersensitivity (DTH)

8-10×10⁶ PBMC cells, along with 0.25LF unit of PBS-dialyzed TetanusToxoid (TT, Kuo Kwang, Cat #K4103-11) or PBS, were injected in a finalvolume of 50 μl into the hind footpad of female B6 mice. Mice of 6-8weeks were used in all experiments. Footpad thickness was measuredbefore and 24 hrs post injection using a dial thickness gauge. Thepre-injected value was subtracted from post-injection value to obtainthe net paw thickness. All measurement values were recorded inmillimeters (mm). h15A7H and h15A7H tetravalent antibodies titrated inPBS were intravenously administered at indicated doses into B6 mice onehour prior to PBMC and TT injection. PBS was used as the vehiclecontrol. 2 or 4 paws (1 or 2 mice) per treatment were tested. The plasmasamples were collected 24 hrs after Ab administration to check theconcentrations of antibody variants. The percent inhibition of pawthickness was calculated as follows: 100×(Δ paw thicknesss_(veh)−Δ pawthickness_(Ab))/(Δ paw thickness_(veh)−Δ paw thickness_(PBMC only)).

ELISA for Detecting Antibody Concentration in Mouse Plasma

96-well microtiter plates were coated with anti-idiotype antibody 12H5.5at 0.5 μg/mL in ELISA coating buffer (30 mM Na₂CO₃/100 mM NaHCO₃) at 4°C. overnight. Plates were then blocked with 200 μL/well of 0.5% BSA inPBS for 1 hour at room temperature, and washed 3 times with ELISAwashing buffer (0.05% Tween20 in PBS), followed by addition of 50μL/well of calibration standard or samples. Calibration standards at aserial dilution were first prepared in the normal mouse plasma.Calibration standard or samples were pre-diluted 1000× in assay diluent(0.1% BSA and 0.05% Tween 20 in PBS), to make a final concentration of0.1% mouse plasma in assay diluents, before dispensing onto the plates.Subsequent dilutions, if needed, were prepared using assay diluentscontaining 0.1% normal mouse plasma. After 1 hour incubation at roomtemperature and washing 5 times with ELISA washing buffer, the secondaryantibody mouse anti-human IgG4 pFc′-HRP (SouthernBiotech Cat. no.9190-05; dilution 1:15000) was added at 50 μL/well and incubated at roomtemperature for 1 hour. The plates were then washed 5 times with ELISAwashing buffer, followed by addition of TMB substrate for colordevelopment. Reactions were stopped by 0.5N H2504, and an absorbancevalue was measured at 450 nm in a microtiter plate reader (MolecularDevice VERSAmax).

Results

Reducing and Non-Reducing SDS-PAGE of Humanized 15A7H TetravalentAntibodies

As shown in FIGS. 2A-2C, SDS-PAGE followed by Coomassie blue stainingwas used to verify the molecular weight and basic structure ofanti-PSGL-1 tetravalent antibodies under non-reducing and reducingconditions. h15A7H V2-V3, V4-V2 and LH10-g4pFc under non-reducingconditions yielded a major protein band with a molecular weight ofaround 150 kDa (FIG. 2A). In the same conditions, h15A7H scFv₂-LC IgG4p,LC-scFv₂IgG4p, and scFv₄-crIgG4p yielded a major protein band with amolecular weight of around 200 kDa (FIG. 2B).

Under reducing conditions, h15A7H V2-V3, V4-V2 and LH10 g4pFc showed asingle band with the expected molecular weight of around 75 kDa, whereasboth h15A7H scFv₂-LC and LC-scFv₂ showed two major bands with similarmolecular weight around 50 kDa (FIG. 2C). One band was the scFv-LC orLC-scFv fusion protein, and the other was the wild type h15A7H heavychain. scFv₄-crIgG4p also showed two major bands, one representing thescFv-CH₁-hinge-CH2-CH3 (around 62.5 kDa) fusion protein, and the otherthe scFv-kappa-fusion (around 37.5 kDa) protein (FIG. 2C). As control,the h15A7H gave a single band with an expected molecular weight of 150kDa in the non-reducing gels (FIGS. 2A & 2B) and two major bands (heavychain: 50 kDa, light chain: 25 kDa) under reducing conditions (FIG. 2C).

Binding of Humanized 15A7H Tetravalent Antibody Variants toSP2/O-hPSGL-1 and SP2/O

The binding ability of h15A7H tetravalent antibodies was evaluated inhuman PSGL-1 SP2/O cells. The h15A7H tetravalent antibody boundpositively to the SP2/O-hPSGL-1, but not to parental SP2/O cell lackingof hPSGL-1 antigen (Table A below). Additionally, wild type h15A7H andall of h15A7H tetravalent antibodies gave similar binding activity onSP2/O-hPSGL-1 (Table A). These results demonstrated that h15A7Htetravalent antibodies retained binding reactivity to hPSGL-1 molecule.

TABLE A Binding activity (measured by mean florescence intensity) ofhumanized 15A7H tetravalent antibodies to SP2/O-hPSGL-1 and SP2/O.SP2/O-hPSGL-1 SP2/O (μg/mL) 3 1 0.3 0.1 3 1 0.3 0.1 h15A7H 4667 64005943 3410 17 26 8 8 h15A7H LH10- 4627 6677 5410 2902 19 18 31 30 g4pFch15A7H V2-V3- 4535 6260 5731 3156 33 22 26 17 g4pFc h15A7H scFv₂- 43825744 7060 5543 24 12 11 24 LC-IgG4p h15A7H V4-V2- 4923 6779 6454 3953 2320 21 14 g4pFc h15A7H scFv₄- 5938 6013 4637 2640 30 28 18 8 crIgG4ph15A7H LC- 6026 5822 3477 3042 24 23 25 3 scFv₂-IgG4p hIgG4p (control)28 ND ND ND 33 ND ND ND ND: not done

In Vitro Apoptosis of SP2/O-hPSGL-1 Cells Induced by Humanized 15A7HTetravalent Antibodies

Induction of apoptosis was evaluated by staining of Annexin V and/or PIin SP2/O-hPSGL-1 cells after incubation with h15A7H or tetravalentantibody. As shown in Table B below, the parental antibody, h15A7H, didnot induce apoptosis in SP2/O-hPSGL-1 cells at the tested concentrationof 0.5 and 0.0625 μg/mL in the absence of cross-linker. At the testedconcentrations of 0.5 μg/mL, all of the h15A7H tetravalent antibodiesinduced apoptosis (ranging from 18-36%). At the lowest testedconcentration tested (0.0625 μg/mL), 3 out of 6 tetravalent h15A7Hantibodies, LH10-g4pFc, V2-V3-g4pFc, and scFv₂-LC-IgG4p, inducedapoptosis in 12-16% of cells, whereas h15A7H V4-V2-g4pFc, scFv₄-crIgG4p,and LC-scFv₂-IgG4p did not induce cell death in SP2/0-hPSGL-1 at thislower dose. These data clearly demonstrate that all of the h15A7Htetravalent antibodies possess apoptosis-inducing ability, but that sometetravalent antibodies do so with greater potency.

TABLE B In vitro apoptosis of SP2/O-hPSGL-1 cells induced by humanized15A7H tetravalent antibodies. Apoptosis % (substrate background, 0.5μg/mL 0.0625 μg/mL n = 4) mean SD mean SD h15A7H 2.75 3.95 1.5 3.32h15A7H LH10-g4pFc 26.75 11.32 11.75* 5.50 h15A7H V2-V3-g4pFc 26.5 5.6913.5* 5.45 h15A7H scFv₂-LC-IgG4p 23.75 9.00 15.5* 5.69 h15A7HV4-V2-g4pFc 30 4.55 0.5 3.00 h15A7H scFv₄-crIgG4p 35.75 7.63 1.75 2.87h15A7H LC-scFv₂-IgG4p 18 9.83 0.5 4.20 SD: standard deviation *T-test Pvalue < 0.05 (compared to treatment with V4-V2-g4pFc, scFv₄-crIgG4p andLC-scFv₂-IgG4p).Efficacy of h15A7H and h15A7H Tetravalent Antibodies in the Inhibitionof Trans-Vivo DTH Response in B6 Mice

The h15A7H and h15A7H tetravalent antibodies described above were testedfor their efficacy in the inhibition of trans vivo DTH response in B6mice. h15A7H antibody was intravenously injected into mice at the dosesof 10 and 1 mg/kg, whereas tetravalent antibodies were intravenouslyinjected into mice at the doses of 1 and 0.3 mg/kg. Experiments wereconducted using PBMCs from four different donors, and % inhibition wascalculated to evaluate the in vivo inhibitory efficacy.

As shown in Table C below, h15A7H antibody could inhibit footpadswelling by a mean of 93% at the dose of 10 mg/kg. The inhibition effectwas reduced to 23% at the low dose of 1 mg/kg. As for 15A7H tetravalentantibodies, variants such as h15A7H LH10-g4p Fc, V2-V3-g4pFc andscFv₂-LC-IgG4p remained effective in inhibition even at doses of 1 or0.3 mg/kg (with 59-76% inhibition).

TABLE C Effect of h15A7H and h15A7H tetravalent antibodies on Trans-vivoDTH. Exp. 1 Exp. 2 Exp. 3 Exp. 4 Mean SEM % inhibition at 10 mg/kgh15A7H 71 104  82 116 93 10.2 % inhibition at 1 mg/kg h15A7H 15 28 18 3223 4.0 h15A7H LH10-g4pFc 75 51 109 69 76 12.1 h15A7H V2-V3-g4pFc 29 33100 91 63 18.7 h15A7H scFv₂-LC-IgG4p 53 34 104 93 71 16.3 h15A7HV4-V2-g4pFc ND ND 11 44 27 16.5 h15A7H scFv₄-crIgG4p 14  2 −18 6 1 6.8h15A7H LC-scFv₂-IgG4p −17  16 14 29 10 9.8 % inhibition at 0.3 mg/kgh15A7H LH10-g4pFc 28 73 109 50 65 17.2 h15A7H V2-V3-g4pFc 24 47 127 8069 22.3 h15A7H scFv₂-LC-IgG4p 74 24 66 73 59 11.8 h15A7H V4-V2-g4pFc NDND −16 11 −2 13.7 h15A7H scFv₄-crIgG4p  2  9 −7 6 3 3.6 h15A7HLC-scFv2-IgG4p −4 19 5 15 9 5.2 ND: not done.; SEM: the standard errorof the mean

Plasma levels of h15A7H and h15A7H tetravalent antibodies were alsomeasured 24 hrs after i.v. administration (Table D). All of theantibodies showed plasma levels around 6513-9025 ng/mL at 1 mg/kg exceptfor V4-V2-g4pFc, which was undetectable after 24 hrs circulation invivo. Without wishing to be bound by theory, it is thought that theseresults may indicate that the difference in efficacy among h15A7H andtetravalent variants could be mainly due to the differences inapoptosis-inducing ability, as demonstrated in Table B.

TABLE D Plasma concentrations of h15A7H and h15A7H tetravalentantibodies in B6 mice. Exp. 1 Exp. 2 Exp. 3 Exp. 4 Mean SEM Conc.(ng/mL) at 10 mg/kg h15A7H 103411  100189  104471  110820  104723  2227 Conc. (ng/mL) at 1 mg/kg h15A7H 9087 8578 8316 10118  9025 398 h15A7HLH10-g4pFc 5698 7333 6335 6686 6513 508 h15A7H V2-V3-g4pFc 6488 81736982 6478 7030 576 h15A7H scFv₂-LC-IgG4p 5766 7082 7452 5979 6570 786h15A7H V4-V2-g4pFc — — BLQ BLQ — — (<100) (<100) h15A7H scFv₄-crIgG4p6156 6924 5997 6353 6358 292 h15A7H LC-scFv₂-IgG4p 7323 8432 9014 90068444 535 Conc. (ng/mL) at 0.3 mg/kg h15A7H LH10-g4pFc 1419 1853 19061793 1743 160 h15A7H V2-V3-g4pFc 1567 2284 2202 2065 2029 239 h15A7HscFv₂-LC-IgG4p 1344 1968 2112 1632 1764 241 h15A7H V4-V2-g4pFc — — BLQBLQ — — (<100) (<100) h15A7H scFv₄-crIgG4p 1256 1909 1772 1668 1651 205h15A7H LC-scFv₂-IgG4p 1765 2493 2325 2356 2235 225 BLQ: beneath limit ofquantification; SEM: the standard error of the mean

In summary, these data demonstrate that various h15A7H tetravalentantibodies possess differential abilities in induction of apoptosis invitro, which correlate with differential abilities in the inhibition ofa DTH response in trans vivo DTH murine model. Those tetravalentantibodies with higher potency for apoptosis induction showed enhancedefficacy compared to h15A7H in the trans-vivo DTH model. These resultssuggest that some of these h15A7H tetravalent variants may havepotential advantages over h15A7H for further clinical development.

Although the foregoing embodiments have been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the present disclosure.

Sequences

All polypeptide sequences are presented N-terminal to C-terminal unlessotherwise noted. All polynucleotide sequences are presented 5′ to 3′unless otherwise noted. The three CDRs in each chain are underlined, andthe linker regions are shown in lower case letters.

Amino acid sequence of h15A7H LH10-g4pFc (SEQ ID NO: 1)DIQMTQSPSSLSASVGDRVTITCRSSQSIVHNDGNTYFEWYQQKPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTHFTLTISSLQPEDFATYYCFQGSYVPLTFGQGTKVEIKggggsggggsEVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYINGGSSTIFYANAVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARYASYGGGAMDYWGQGTLVTVSSggggsggggsggggsggggsggggsDIQMTQSPSSLSASVGDRVTITCRSSQSIVHNDGNTYFEWYQQKPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTHFTLTISSLQPEDFATYYCFQGSYVPLTFGQGTKVEIKggggsggggsEVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYINGGSSTIFYANAVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARYASYGGGAMDYWGQGTLVTVSSggggsaaaESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKcDNA sequence of h15A7H LH10-g4pFc (SEQ ID NO: 2)GACATTCAGATGACCCAATCTCCGAGCTCTTTGTCTGCGTCTGTAGGGGATAGGGTCACTATCACCTGCAGATCTAGTCAGAGCATTGTACATAATGATGGAAACACCTATTTTGAATGGTACCAACAGAAACCAGGAAAGGCACCCAAGCTTCTCATCTATAAAGTTTCCAATCGATTTTCTGGTGTCCCATCCAGGTTTAGTGGCAGTGGGTCTGGGACACACTTCACCCTCACCATCTCTTCTCTGCAGCCGGAGGATTTCGCAACCTATTACTGTTTTCAAGGTTCATATGTTCCTCTCACGTTCGGTCAAGGCACCAAGGTGGAAATCAAAggtggaggcggttcaggcggaggtggctctGAAGTGCAACTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGAAGCTTGAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTTTGGAATGCACTGGGTTCGCCAGGCTCCAGGGAAGGGACTCGAGTGGGTCGCATACATTAATGGTGGCAGTAGTACCATCTTCTATGCAAACGCAGTGAAGGGCCGATTCACCATCTCCAGAGATAATGCCAAGAACACCCTGTACCTGCAAATGAATTCTCTGAGGGCTGAGGACACGGCCGTGTATTACTGTGCAAGATATGCTAGTTACGGAGGGGGTGCTATGGACTATTGGGGCCAAGGCACCCTGGTCACAGTCTCCTCAggtggaggcggttcaggcggaggtggctctggcggtggcggatccggaggcggaggttccggaggtggcggaagtGACATTCAGATGACCCAATCTCCGAGCTCTTTGTCTGCGTCTGTAGGGGATAGGGTCACTATCACCTGCAGATCTAGTCAGAGCATTGTACATAATGATGGAAACACCTATTTTGAATGGTACCAACAGAAACCAGGAAAGGCACCCAAGCTTCTCATCTATAAAGTTTCCAATCGATTTTCTGGTGTCCCATCCAGGTTTAGTGGCAGTGGGTCTGGGACACACTTCACCCTCACCATCTCTTCTCTGCAGCCGGAGGATTTCGCAACCTATTACTGTTTTCAAGGTTCATATGTTCCTCTCACGTTCGGTCAAGGCACCAAGGTGGAAATCAAAggtggaggcggttcaggcggaggtggctctGAAGTGCAACTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGAAGCTTGAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTTTGGAATGCACTGGGTTCGCCAGGCTCCAGGGAAGGGACTCGAGTGGGTCGCATACATTAATGGTGGCAGTAGTACCATCTTCTATGCAAACGCAGTGAAGGGCCGATTCACCATCTCCAGAGATAATGCCAAGAACACCCTGTACCTGCAAATGAATTCTCTGAGGGCTGAGGACACGGCCGTGTATTACTGTGCAAGATATGCTAGTTACGGAGGGGGTGCTATGGACTATTGGGGCCAAGGCACCCTGGTCACAGTCTCCTCAggaggcggaggttccgcggccgcaGAGTCCAAATATGGTCCCCCATGCCCACCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAATGA Amino acid sequence of h15A7H V2-V3-g4pFc (SEQ ID NO: 3)EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKCLEWVAYINGGSSTIFYANAVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARYASYGGGAMDYWGQGTLVTVSSggggsggggsggggsggggsggggsDIQMTQSPSSLSASVGDRVTITCRSSQSIVHNDGNTYFEWYQQKPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTHFTLTISSLQPEDFATYYCFQGSYVPLTFGCGTKVEIKastgsDIQMTQSPSSLSASVGDRVTITCRSSQSIVHNDGNTYFEWYQQKPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTHFTLTISSLQPEDFATYYCFQGSYVPLTFGQGTKVEIKggggsggggsggggsggggsggggsEVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYINGGSSTIFYANAVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARYASYGGGAMDYWGQGTLVTVSSggggsaaaESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKcDNA sequence of h15A7H V2-V3-g4pFc (SEQ ID NO: 4)GAAGTGCAACTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGAAGCTTGAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTTTGGAATGCACTGGGTTCGCCAGGCTCCAGGGAAGTGTCTCGAGTGGGTCGCATACATTAATGGTGGCAGTAGTACCATCTTCTATGCAAACGCAGTGAAGGGCCGATTCACCATCTCCAGAGATAATGCCAAGAACACCCTGTACCTGCAAATGAATTCTCTGAGGGCTGAGGACACGGCCGTGTATTACTGTGCAAGATATGCTAGTTACGGAGGGGGTGCTATGGACTATTGGGGCCAAGGCACCCTGGTCACAGTCTCCTCAggtggaggcggttcaggcggaggtggctctggcggtggcggatccggaggcggaggttccggaggtggcggaagtGACATTCAGATGACCCAATCTCCGAGCTCTTTGTCTGCGTCTGTAGGGGATAGGGTCACTATCACCTGCAGATCTAGTCAGAGCATTGTACATAATGATGGAAACACCTATTTTGAATGGTACCAACAGAAACCAGGAAAGGCACCCAAGCTTCTCATCTATAAAGTTTCCAATCGATTTTCTGGTGTCCCATCCAGGTTTAGTGGCAGTGGGTCTGGGACACACTTCACCCTCACCATCTCTTCTCTGCAGCCGGAGGATTTCGCAACCTATTACTGTTTTCAAGGTTCATATGTTCCTCTCACGTTCGGTTGTGGCACCAAGGTGGAAATCAAAgcttcaaccggttcaGACATTCAGATGACCCAATCTCCGAGCTCTTTGTCTGCGTCTGTAGGGGATAGGGTCACTATCACCTGCAGATCTAGTCAGAGCATTGTACATAATGATGGAAACACCTATTTTGAATGGTACCAACAGAAACCAGGAAAGGCACCCAAGCTTCTCATCTATAAAGTTTCCAATCGATTTTCTGGTGTCCCATCCAGGTTTAGTGGCAGTGGGTCTGGGACACACTTCACCCTCACCATCTCTTCTCTGCAGCCGGAGGATTTCGCAACCTATTACTGTTTTCAAGGTTCATATGTTCCTCTCACGTTCGGTCAAGGCACCAAGGTGGAAATCAAAggtggaggcggttcaggcggaggtggctctggcggtggcggatccggaggcggaggttccggaggtggcggaagtGAAGTGCAACTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGAAGCTTGAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTTTGGAATGCACTGGGTTCGCCAGGCTCCAGGGAAGGGACTCGAGTGGGTCGCATACATTAATGGTGGCAGTAGTACCATCTTCTATGCAAACGCAGTGAAGGGCCGATTCACCATCTCCAGAGATAATGCCAAGAACACCCTGTACCTGCAAATGAATTCTCTGAGGGCTGAGGACACGGCCGTGTATTACTGTGCAAGATATGCTAGTTACGGAGGGGGTGCTATGGACTATTGGGGCCAAGGCACCCTGGTCACAGTCTCCTCAggaggcggaggttccgcggccgcaGAGTCCAAATATGGTCCCCCATGCCCACCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAATGAAmino acid sequence of h15A7H V4-V2-g4pFc (SEQ ID NO: 5)DIQMTQSPSSLSASVGDRVTITCRSSQSIVHNDGNTYFEWYQQKPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTHFTLTISSLQPEDFATYYCFQGSYVPLTFGCGTKVEIKggggsggggsggggsggggsggggsEVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKCLEWVAYINGGSSTIFYANAVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARYASYGGGAMDYWGQGTLVTVSSastgsEVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKCLEWVAYINGGSSTIFYANAVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARYASYGGGAMDYWGQGTLVTVSSggggsggggsggggsggggsggggsDIQMTQSPSSLSASVGDRVTITCRSSQSIVHNDGNTYFEWYQQKPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTHFTLTISSLQPEDFATYYCFQGSYVPLTFGCGTKVEIKggggsaaaESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKcDNA sequence of h15A7H V4-V2-g4pFc (SEQ ID NO: 6)GACATTCAGATGACCCAATCTCCGAGCTCTTTGTCTGCGTCTGTAGGGGATAGGGTCACTATCACCTGCAGATCTAGTCAGAGCATTGTACATAATGATGGAAACACCTATTTTGAATGGTACCAACAGAAACCAGGAAAGGCACCCAAGCTTCTCATCTATAAAGTTTCCAATCGATTTTCTGGTGTCCCATCCAGGTTTAGTGGCAGTGGGTCTGGGACACACTTCACCCTCACCATCTCTTCTCTGCAGCCGGAGGATTTCGCAACCTATTACTGTTTTCAAGGTTCATATGTTCCTCTCACGTTCGGTTGTGGCACCAAGGTGGAAATCAAAggtggaggcggttcaggcggaggtggctctggcggtggcggatccggaggcggaggttccggaggtggcggaagtGAAGTGCAACTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGAAGCTTGAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTTTGGAATGCACTGGGTTCGCCAGGCTCCAGGGAAGTGTCTCGAGTGGGTCGCATACATTAATGGTGGCAGTAGTACCATCTTCTATGCAAACGCAGTGAAGGGCCGATTCACCATCTCCAGAGATAATGCCAAGAACACCCTGTACCTGCAAATGAATTCTCTGAGGGCTGAGGACACGGCCGTGTATTACTGTGCAAGATATGCTAGTTACGGAGGGGGTGCTATGGACTATTGGGGCCAAGGCACCCTGGTCACAGTCTCCTCAgcttcaaccggttcaGAAGTGCAACTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGAAGCTTGAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTTTGGAATGCACTGGGTTCGCCAGGCTCCAGGGAAGTGTCTCGAGTGGGTCGCATACATTAATGGTGGCAGTAGTACCATCTTCTATGCAAACGCAGTGAAGGGCCGATTCACCATCTCCAGAGATAATGCCAAGAACACCCTGTACCTGCAAATGAATTCTCTGAGGGCTGAGGACACGGCCGTGTATTACTGTGCAAGATATGCTAGTTACGGAGGGGGTGCTATGGACTATTGGGGCCAAGGCACCCTGGTCACAGTCTCCTCAggtggaggcggttcaggcggaggtggctctggcggtggcggatccggaggcggaggttccggaggtggcggaagtGACATTCAGATGACCCAATCTCCGAGCTCTTTGTCTGCGTCTGTAGGGGATAGGGTCACTATCACCTGCAGATCTAGTCAGAGCATTGTACATAATGATGGAAACACCTATTTTGAATGGTACCAACAGAAACCAGGAAAGGCACCCAAGCTTCTCATCTATAAAGTTTCCAATCGATTTTCTGGTGTCCCATCCAGGTTTAGTGGCAGTGGGTCTGGGACACACTTCACCCTCACCATCTCTTCTCTGCAGCCGGAGGATTTCGCAACCTATTACTGTTTTCAAGGTTCATATGTTCCTCTCACGTTCGGTTGTGGCACCAAGGTGGAAATCAAAggaggcggaggttccgcggccgcaGAGTCCAAATATGGTCCCCCATGCCCACCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAATGAAmino acid sequence of h15A7H scFv₂-LC-IgG4p Light chain (SEQ ID NO: 7)EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKCLEWVAYINGGSSTIFYANAVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARYASYGGGAMDYWGQGTLVTVSSggggsggggsggggsggggsggggsDIQMTQSPSSLSASVGDRVTITCRSSQSIVHNDGNTYFEWYQQKPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTHFTLTISSLQPEDFATYYCFQGSYVPLTFGCGTKVEIKastgsggggsDIQMTQSPSSLSASVGDRVTITCRSSQSIVHNDGNTYFEWYQQKPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTHFTLTISSLQPEDFATYYCFQGSYVPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECcDNA sequence of h15A7H scFv₂-LC-IgG4p Light chain (SEQ ID NO: 8)GAAGTGCAACTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGAAGCTTGAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTTTGGAATGCACTGGGTTCGCCAGGCTCCAGGGAAGTGTCTCGAGTGGGTCGCATACATTAATGGTGGCAGTAGTACCATCTTCTATGCAAACGCAGTGAAGGGCCGATTCACCATCTCCAGAGATAATGCCAAGAACACCCTGTACCTGCAAATGAATTCTCTGAGGGCTGAGGACACGGCCGTGTATTACTGTGCAAGATATGCTAGTTACGGAGGGGGTGCTATGGACTATTGGGGCCAAGGCACCCTGGTCACAGTCTCCTCAggtggaggcggttcaggcggaggtggctctggcggtggcggatccggaggcggaggttccggaggtggcggaagtGACATTCAGATGACCCAATCTCCGAGCTCTTTGTCTGCGTCTGTAGGGGATAGGGTCACTATCACCTGCAGATCTAGTCAGAGCATTGTACATAATGATGGAAACACCTATTTTGAATGGTACCAACAGAAACCAGGAAAGGCACCCAAGCTTCTCATCTATAAAGTTTCCAATCGATTTTCTGGTGTCCCATCCAGGTTTAGTGGCAGTGGGTCTGGGACACACTTCACCCTCACCATCTCTTCTCTGCAGCCGGAGGATTTCGCAACCTATTACTGTTTTCAAGGTTCATATGTTCCTCTCACGTTCGGTTGTGGCACCAAGGTGGAAATCAAAgcttcaaccggttcaggaggtggcggaagtGACATTCAGATGACCCAATCTCCGAGCTCTTTGTCTGCGTCTGTAGGGGATAGGGTCACTATCACCTGCAGATCTAGTCAGAGCATTGTACATAATGATGGAAACACCTATTTTGAATGGTACCAACAGAAACCAGGAAAGGCACCCAAGCTTCTCATCTATAAAGTTTCCAATCGATTTTCTGGTGTCCCATCCAGGTTTAGTGGCAGTGGGTCTGGGACACACTTCACCCTCACCATCTCTTCTCTGCAGCCGGAGGATTTCGCAACCTATTACTGTTTTCAAGGTTCATATGTTCCTCTCACGTTCGGTCAAGGCACCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGAmino acid sequence of h15A7H LC- scFv₂-IgG4p light chain (SEQ ID NO: 9)DIQMTQSPSSLSASVGDRVTITCRSSQSIVHNDGNTYFEWYQQKPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTHFTLTISSLQPEDFATYYCFQGSYVPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECggggsggggsEVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKCLEWVAYINGGSSTIFYANAVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARYASYGGGAMDYWGQGTLVTVSSggggsggggsggggsggggsggggsDIQMTQSPSSLSASVGDRVTITCRSSQSIVHNDGNTYFEWYQQKPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTHFTLTISSLQPEDFATYYCFQGSYVPLTFGCGTKVEIKAAAHHHHHHHHHHcDNA sequence of h15A7H LC- scFv₂-IgG4p light chain (SEQ ID NO: 10)GACATTCAGATGACCCAATCTCCGAGCTCTTTGTCTGCGTCTGTAGGGGATAGGGTCACTATCACCTGCAGATCTAGTCAGAGCATTGTACATAATGATGGAAACACCTATTTTGAATGGTACCAACAGAAACCAGGAAAGGCACCCAAGCTTCTCATCTATAAAGTTTCCAATCGATTTTCTGGTGTCCCATCCAGGTTTAGTGGCAGTGGGTCTGGGACACACTTCACCCTCACCATCTCTTCTCTGCAGCCGGAGGATTTCGCAACCTATTACTGTTTTCAAGGTTCATATGTTCCTCTCACGTTCGGTCAAGGCACCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTggtggaggcggttcaggcggaggtggctctGAAGTGCAACTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGAAGCTTGAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTTTGGAATGCACTGGGTTCGCCAGGCTCCAGGGAAGTGTCTCGAGTGGGTCGCATACATTAATGGTGGCAGTAGTACCATCTTCTATGCAAACGCAGTGAAGGGCCGATTCACCATCTCCAGAGATAATGCCAAGAACACCCTGTACCTGCAAATGAATTCTCTGAGGGCTGAGGACACGGCCGTGTATTACTGTGCAAGATATGCTAGTTACGGAGGGGGTGCTATGGACTATTGGGGCCAAGGCACCCTGGTCACAGTCTCCTCAggtggaggcggttcaggcggaggtggctctggcggtggcggatccggaggcggaggttccggaggtggcggaagtGACATTCAGATGACCCAATCTCCGAGCTCTTTGTCTGCGTCTGTAGGGGATAGGGTCACTATCACCTGCAGATCTAGTCAGAGCATTGTACATAATGATGGAAACACCTATTTTGAATGGTACCAACAGAAACCAGGAAAGGCACCCAAGCTTCTCATCTATAAAGTTTCCAATCGATTTTCTGGTGTCCCATCCAGGTTTAGTGGCAGTGGGTCTGGGACACACTTCACCCTCACCATCTCTTCTCTGCAGCCGGAGGATTTCGCAACCTATTACTGTTTTCAAGGTTCATATGTTCCTCTCACGTTCGGTTGTGGCACCAAGGTGGAAATCAAAGCGGCCGCACATCATCATCATCATCACCACCACCACCACTAGAmino acid sequence of h15A7H scFv₂-LC-IgG4p and h15A7 LC- ScFv₂ -IgG4pheavy chain (SEQ ID NO: 11)EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYINGGSSTIFYANAVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARYASYGGGAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKcDNA sequence of h15A7H scFv₂-LC-IgG4p and h15A7 LC- scFv₂ -IgG4p heavy chain(SEQ ID NO: 12)GAAGTGCAACTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGAAGCTTGAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTTTGGAATGCACTGGGTTCGCCAGGCTCCAGGGAAGGGACTCGAGTGGGTCGCATACATTAATGGTGGCAGTAGTACCATCTTCTATGCAAACGCAGTGAAGGGCCGATTCACCATCTCCAGAGATAATGCCAAGAACACCCTGTACCTGCAAATGAATTCTCTGAGGGCTGAGGACACGGCCGTGTATTACTGTGCAAGATATGCTAGTTACGGAGGGGGTGCTATGGACTATTGGGGCCAAGGCACCCTGGTCACAGTCTCCTCAGCTTCCACCAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAATGAAmino acid sequence of h15A7H scFv₄-crIgG4p light chain (SEQ ID NO: 13)EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKCLEWVAYINGGSSTIFYANAVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARYASYGGGAMDYWGQGTLVTVSSggggsggggsggggsggggsggggsDIQMTQSPSSLSASVGDRVTITCRSSQSIVHNDGNTYFEWYQQKPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTHFTLTISSLQPEDFATYYCFQGSYVPLTFGCGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECcDNA sequence of h15A7H scFv₄-crIgG4p light chain (SEQ ID NO: 14)GAAGTGCAACTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGAAGCTTGAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTTTGGAATGCACTGGGTTCGCCAGGCTCCAGGGAAGTGTCTCGAGTGGGTCGCATACATTAATGGTGGCAGTAGTACCATCTTCTATGCAAACGCAGTGAAGGGCCGATTCACCATCTCCAGAGATAATGCCAAGAACACCCTGTACCTGCAAATGAATTCTCTGAGGGCTGAGGACACGGCCGTGTATTACTGTGCAAGATATGCTAGTTACGGAGGGGGTGCTATGGACTATTGGGGCCAAGGCACCCTGGTCACAGTCTCCTCAggtggaggcggttcaggcggaggtggctctggcggtggcggatccggaggcggaggttccggaggtggcggaagtGACATTCAGATGACCCAATCTCCGAGCTCTTTGTCTGCGTCTGTAGGGGATAGGGTCACTATCACCTGCAGATCTAGTCAGAGCATTGTACATAATGATGGAAACACCTATTTTGAATGGTACCAACAGAAACCAGGAAAGGCACCCAAGCTTCTCATCTATAAAGTTTCCAATCGATTTTCTGGTGTCCCATCCAGGTTTAGTGGCAGTGGGTCTGGGACACACTTCACCCTCACCATCTCTTCTCTGCAGCCGGAGGATTTCGCAACCTATTACTGTTTTCAAGGTTCATATGTTCCTCTCACGTTCGGTTGTGGCACCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGAmino acid sequence of h15A7H scFv₄-crIgG4p heavy chain (SEQ ID NO: 15)EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKCLEWVAYINGGSSTIFYANAVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARYASYGGGAMDYWGQGTLVTVSSggggsggggsggggsggggsggggsDIQMTQSPSSLSASVGDRVTITCRSSQSIVHNDGNTYFEWYQQKPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTHFTLTISSLQPEDFATYYCFQGSYVPLTFGCGTKVEIKASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKcDNA sequence of h15A7H scFv₄-crIgG4p heavy chain (SEQ ID NO: 16)GAAGTGCAACTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGAAGCTTGAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTTTGGAATGCACTGGGTTCGCCAGGCTCCAGGGAAGTGTCTCGAGTGGGTCGCATACATTAATGGTGGCAGTAGTACCATCTTCTATGCAAACGCAGTGAAGGGCCGATTCACCATCTCCAGAGATAATGCCAAGAACACCCTGTACCTGCAAATGAATTCTCTGAGGGCTGAGGACACGGCCGTGTATTACTGTGCAAGATATGCTAGTTACGGAGGGGGTGCTATGGACTATTGGGGCCAAGGCACCCTGGTCACAGTCTCCTCAggtggaggcggttcaggcggaggtggctctggcggtggcggatccggaggcggaggttccggaggtggcggaagtGACATTCAGATGACCCAATCTCCGAGCTCTTTGTCTGCGTCTGTAGGGGATAGGGTCACTATCACCTGCAGATCTAGTCAGAGCATTGTACATAATGATGGAAACACCTATTTTGAATGGTACCAACAGAAACCAGGAAAGGCACCCAAGCTTCTCATCTATAAAGTTTCCAATCGATTTTCTGGTGTCCCATCCAGGTTTAGTGGCAGTGGGTCTGGGACACACTTCACCCTCACCATCTCTTCTCTGCAGCCGGAGGATTTCGCAACCTATTACTGTTTTCAAGGTTCATATGTTCCTCTCACGTTCGGTTGTGGCACCAAGGTGGAAATCAAAGCTTCCACCAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAATGAAmino acid sequence of h15A7H CDR-H1 (SEQ ID NO: 17) SFGMHAmino acid sequence of h15A7H CDR-H2 (SEQ ID NO: 18) YINGGSSTIFYANAVKGAmino acid sequence of h15A7H CDR-H3 (SEQ ID NO: 19) YASYGGGAMDYAmino acid sequence of h15A7H CDR-L1 (SEQ ID NO: 20) RSSQSIVHNDGNTYFEAmino acid sequence of h15A7H CDR-L2 (SEQ ID NO: 21) KVSNRFSAmino acid sequence of h15A7H CDR-L3 (SEQ ID NO: 22) FQGSYVPLTAmino acid sequence of h15A7H VH (SEQ ID NO: 23)EVQLVESGGGLVQPGGSLRLSCAASGETFSSFGMHWVRQAPGKGLEWVAYINGGSSTIFYANAVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARYASYGGGAMDYWGQGTLVTVSSAmino acid sequence of h15A7H VL (SEQ ID NO: 24)DIQMTQSPSSLSASVGDRVTITCRSSQSIVHNDGNTYFEWYQQKPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTHFTLTISSLQPEDFATYYCFQGSYVPLTFGQGTKVEIKAmino acid sequence of linker sequence repeat (SEQ ID NO: 25) ggggsAmino acid sequence of linker with Fc (SEQ ID NO: 26) ggggsaaaAmino acid sequence of taFv linker (SEQ ID NO: 27) astgsAmino acid sequence of scFv light chain linker (SEQ ID NO: 28)astgsggggs Amino acid sequence of h15A7H VH G44C (SEQ ID NO: 29)EVQLVESGGGLVQPGGSLRLSCAASGETFSSFGMHWVRQAPGKCLEWVAYINGGSSTIFYANAVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARYASYGGGAMDYWGQGTLVTVSSAmino acid sequence of h15A7H VL Q100C (SEQ ID NO: 30)DIQMTQSPSSLSASVGDRVTITCRSSQSIVHNDGNTYFEWYQQKPGKAPKLLIYKVSNRFSGVPSRFSGSGSGTHFTLTISSLQPEDFATYYCFQGSYVPLTFGCGTKVEIK Amino acid sequence of human PSGL-1(SEQ ID NO: 31)MPLQLLLLLILLGPGNSLQLWDTWADEAEKALGPLLARDRRQATEYEYLDYDFLPETEPPEMLRNSTDTTPLTGPGTPESTTVEPAARRSTGLDAGGAVTELTTELANMGNLSTDSAAMEIQTTQPAATEAQTTQPVPTEAQTTPLAATEAQTTRLTATEAQTTPLAATEAQTTPPAATEAQTTQPTGLEAQTTAPAAMEAQTTAPAAMEAQTTPPAAMEAQTTQTTAMEAQTTAPEATEAQTTQPTATEAQTTPLAAMEALSTEPSATEALSMEPTTKRGLFIPFSVSSVTHKGIPMAASNLSVNYPVGAPDHISVKQCLLAILILALVATIFFVCTVVLAVRLSRKGHMYPVRNYSPTEMVCISSLLPDGGEGPSATANGGLSKAKSPGLTPEPREDREGDDLTLHSFLPAmino acid sequence of shorter human PSGL-1 variant (SEQ ID NO: 32)MPLQLLLLLILLGPGNSLQLWDTWADEAEKALGPLLARDRRQATEYEYLDYDFLPETEPPEMLRNSTDTTPLTGPGTPESTTVEPAARRSTGLDAGGAVTELTTELANMGNLSTDSAAMEIQTTQPAATEAQTTPLAATEAQTTRLTATEAQTTPLAATEAQTTPPAATEAQTTQPTGLEAQTTAPAAMEAQTTAPAAMEAQTTPPAAMEAQTTQTTAMEAQTTAPEATEAQTTQPTATEAQTTPLAAMEALSTEPSATEALSMEPTTKRGLFIPFSVSSVTHKGIPMAASNLSVNYPVGAPDHISVKQCLLAILILALVATIFFVCTVVLAVRLSRKGHMYPVRNYSPTEMVCISSLLPDGGEGPSATANGGLSKAKSPGLTPEPREDREGDDLTLHSFLP

1. A tetravalent antibody that specifically binds to human PSGL-1, thetetravalent antibody comprising a dimer of two monomers, wherein eachmonomer of the dimer comprises a single-chain polypeptide comprising,from N-terminus to C-terminus: (a) a first light chain variable (VL)domain; (b) a first linker sequence; (c) a first heavy chain variable(VH) domain; (d) a second linker sequence; (e) a second VL domain; (f) athird linker sequence; (g) a second VH domain; (h) a fourth linkersequence; and (i) an antibody Fc domain, wherein each of the first andthe second VL domains comprises a CDR-L1, a CDR-L2, and a CDR-L3;wherein each of the first and the second VH domains comprises a CDR-H1,a CDR-H2, and a CDR-H3; and wherein each of the first and the second VLdomains forms a VH-VL binding unit with a corresponding VH domain of thefirst and the second VH domains, and wherein each of the two VH-VLbinding units is specific for human PSGL-1.
 2. The tetravalent antibodyof claim 1, wherein at least one of the two VH domains comprises: (i) aCDR-H1 comprising the amino acid sequence of SEQ ID NO:17; (ii) a CDR-H2comprising the amino acid sequence of SEQ ID NO:18; and (iii) a CDR-H3comprising the amino acid sequence of SEQ ID NO:19; and/or wherein atleast one of the two VL domains comprises: (i) a CDR-L1 comprising theamino acid sequence of SEQ ID NO:20; (ii) a CDR-L2 comprising the aminoacid sequence of SEQ ID NO:21; and (iii) a CDR-L3 comprising the aminoacid sequence of SEQ ID NO:22.
 3. (canceled)
 4. The tetravalent antibodyof claim 2, wherein each of the two VH domains comprises the amino acidsequence of SEQ ID NO:23; an amino acid sequence having at least 90%, atleast 95%, or at least 99% sequence identity to SEQ ID NO:23; the aminoacid sequence of SEQ ID NO:29; or an amino acid sequence having at least90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:29;and/or wherein each of the two VL domains comprises the amino acidsequence of SEQ ID NO:24; an amino acid sequence having at least 90%, atleast 95%, or at least 99% sequence identity to SEQ ID NO:24; the aminoacid sequence of SEQ ID NO:30; or an amino acid sequence having at least90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:30.5-9. (canceled)
 10. The tetravalent antibody of claim 1, wherein thefirst, second and third linker sequences each comprise two or morerepeats of the amino acid sequence of SEQ ID NO:25, or the first, secondor third linker sequence comprises the amino acid sequence of SEQ IDNO:33, 34, 35, or
 36. 11. (canceled)
 12. (canceled)
 13. The tetravalentantibody of claim 1, wherein the fourth linker sequence comprises theamino acid sequence of SEQ ID NO:26.
 14. The tetravalent antibody ofclaim 1, wherein each of the two single-chain polypeptides comprises theamino acid sequence of SEQ ID NO:1, or an amino acid sequence having atleast 90%, at least 95%, or at least 99% sequence identity to SEQ IDNO:1.
 15. (canceled)
 16. A tetravalent antibody that specifically bindsto human PSGL-1, the tetravalent antibody comprising a dimer of twomonomers, wherein each monomer of the dimer comprises a single-chainpolypeptide comprising, from N-terminus to C-terminus: (a) a first heavychain variable (VH) domain; (b) a first linker sequence; (c) a firstlight chain variable (VL) domain; (d) a second linker sequence; (e) asecond VL domain; (f) a third linker sequence; (g) a second VH domain;(h) a fourth linker sequence; and (i) an antibody Fc domain, whereineach of the first and the second VL domains comprises a CDR-L1, aCDR-L2, and a CDR-L3; wherein each of the first and the second VHdomains comprises a CDR-H1, a CDR-H2, and a CDR-H3; and wherein each ofthe first and the second VL domains forms a VH-VL binding unit with acorresponding VH domain of the first and the second VH domains, andwherein each of the two VH-VL binding units is specific for humanPSGL-1.
 17. The tetravalent antibody of claim 16, wherein at least oneof the two VH domains comprises: (i) a CDR-H1 comprising the amino acidsequence of SEQ ID NO:17; (ii) a CDR-H2 comprising the amino acidsequence of SEQ ID NO:18; and (iii) a CDR-H3 comprising the amino acidsequence of SEQ ID NO:19; and/or wherein at least one of the two VLdomains comprises: (i) a CDR-L1 comprising the amino acid sequence ofSEQ ID NO:20; (ii) a CDR-L2 comprising the amino acid sequence of SEQ IDNO:21; and (iii) a CDR-L3 comprising the amino acid sequence of SEQ IDNO:22.
 18. (canceled)
 19. The tetravalent antibody of claim 17, whereineach of the two VH domains comprises the amino acid sequence of SEQ IDNO:23; an amino acid sequence having at least 90%, at least 95%, or atleast 99% sequence identity to SEQ ID NO:23; the amino acid sequence ofSEQ ID NO:29; or an amino acid sequence having at least 90%, at least95%, or at least 99% sequence identity to SEQ ID NO:29; and/or whereineach of the two VL domains comprises the amino acid sequence of SEQ IDNO:24; an amino acid sequence having at least 90%, at least 95%, or atleast 99% sequence identity to SEQ ID NO:24; the amino acid sequence ofSEQ ID NO:30; or an amino acid sequence having at least 90%, at least95%, or at least 99% sequence identity to SEQ ID NO:30. 20-24.(canceled)
 25. The tetravalent antibody of claim 16, wherein the firstand the third linker sequences have the same sequence comprising fiverepeats of SEQ ID NO:25.
 26. (canceled)
 27. The tetravalent antibody ofclaim 16, wherein the fourth linker sequence comprises the amino acidsequence of SEQ ID NO:26.
 28. The tetravalent antibody of claim 16,wherein each of the two single-chain polypeptides comprises the aminoacid sequence of SEQ ID NO:3, or an amino acid sequence having at least90%, at least 95%, or at least 99% sequence identity to SEQ ID NO:3. 29.(canceled)
 30. A tetravalent antibody that specifically binds to humanPSGL-1, the tetravalent antibody comprising a dimer of two monomers,wherein each monomer of the dimer comprises an antibody heavy chain andan antibody light chain; wherein the antibody light chain comprises,from N-terminus to C-terminus: (i) a first heavy chain variable (VH)domain, (ii) a first linker sequence, (iii) a first light chain variable(VL) domain, (iv) a second linker sequence, (v) a second VL domain, and(vi) a light chain constant (CL) domain; wherein the antibody heavychain comprises: (i) a second VH domain, and (ii) a heavy chain constantregion comprising a first heavy chain constant region (CH₁) domain, anantibody hinge region, an second heavy chain constant region (CH₂)domain, and a third heavy chain constant region (CH₃) domain; whereineach of the first and the second VL domains comprises a CDR-L1, aCDR-L2, and a CDR-L3; wherein each of the first and the second VHdomains comprises a CDR-H1, a CDR-H2, and a CDR-H3; and wherein each ofthe first and the second VL domains forms a VH-VL binding unit with acorresponding VH domain of the first and the second VH domains, andwherein each of the two VH-VL binding units is specific for humanPSGL-1.
 31. The tetravalent antibody of claim 30, wherein at least oneof the first and the second VH domains comprises: (i) a CDR-H1comprising the amino acid sequence of SEQ ID NO:17; (ii) a CDR-H2comprising the amino acid sequence of SEQ ID NO:18; and (iii) a CDR-H3comprising the amino acid sequence of SEQ ID NO:19; and/or wherein atleast one of the first and the second VL domains comprises: (i) a CDR-L1comprising the amino acid sequence of SEQ ID NO:20; (ii) a CDR-L2comprising the amino acid sequence of SEQ ID NO:21; and (iii) a CDR-L3comprising the amino acid sequence of SEQ ID NO:22.
 32. (canceled) 33.The tetravalent antibody of claim 31, wherein the first and the secondVH domains each comprise the amino acid sequence of SEQ ID NO:23; anamino acid sequence having at least 90%, at least 95%, or at least 99%sequence identity to SEQ ID NO:23; the amino acid sequence of SEQ IDNO:29; or an amino acid sequence having at least 90%, at least 95%, orat least 99% sequence identity to SEQ ID NO:29; and/or wherein each ofthe two VL domains comprises the amino acid sequence of SEQ ID NO:24; anamino acid sequence having at least 90%, at least 95%, or at least 99%sequence identity to SEQ ID NO:24; the amino acid sequence of SEQ IDNO:30; or an amino acid sequence having at least 90%, at least 95%, orat least 99% sequence identity to SEQ ID NO:30. 34-38. (canceled) 39.The tetravalent antibody of claim 30, wherein the CL domain is a kappaCL domain.
 40. The tetravalent antibody of claim 30, wherein the firstlinker sequence comprises five repeats of SEQ ID NO:25; and/or whereinthe second linker sequence comprises the amino acid sequence of SEQ IDNO:28.
 41. (canceled)
 42. The tetravalent antibody of claim 30, whereinthe antibody light chain comprises the amino acid sequence of SEQ IDNO:7, or an amino acid sequence having at least 90%, at least 95%, or atleast 99% sequence identity to SEQ ID NO:7; and/or wherein the antibodyheavy chain comprises the amino acid sequence of SEQ ID NO:11, or anamino acid sequence having at least 90%, at least 95%, or at least 99%sequence identity to SEQ ID NO:11. 43-45. (canceled)
 46. The tetravalentantibody of claim 1, wherein the antibody Fc domain is a human antibodyFc domain. 47-49. (canceled)
 50. An isolated polynucleotide encoding thetetravalent antibody of claim
 1. 51. (canceled)
 52. A vector comprisingthe isolated polynucleotide of claim
 50. 53. A host cell comprising thepolynucleotide of claim
 50. 54. A method of producing a tetravalentantibody comprising culturing the host cell of claim 53 so that thetetravalent antibody is produced.
 55. (canceled)
 56. A pharmaceuticalcomposition comprising the tetravalent antibody of claim 1 and apharmaceutically acceptable carrier. 57-63. (canceled)
 64. A method oftreating a T-cell mediated inflammatory disease, the method comprisingadministering to a subject in need thereof a therapeutically effectiveamount of the tetravalent antibody of claim
 1. 65. A method for treatingan individual in need of a transfusion or transplantation, comprisingadministering to the individual a therapeutically effective amount ofthe tetravalent antibody of claim 1 before, concurrently with, and/orafter the transfusion or transplantation. 66-70. (canceled)